WO2023201786A1 - 一种免疫佐剂组合物和基于该组合物的癌症疫苗及其应用 - Google Patents

一种免疫佐剂组合物和基于该组合物的癌症疫苗及其应用 Download PDF

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WO2023201786A1
WO2023201786A1 PCT/CN2022/092201 CN2022092201W WO2023201786A1 WO 2023201786 A1 WO2023201786 A1 WO 2023201786A1 CN 2022092201 W CN2022092201 W CN 2022092201W WO 2023201786 A1 WO2023201786 A1 WO 2023201786A1
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cpg
odn
components
water
vaccine
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PCT/CN2022/092201
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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
    • A61K39/39—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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/5152—Tumor cells
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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 invention relates to the field of immunotherapy, and in particular to an immune adjuvant composition and a cancer vaccine based on the composition and its application.
  • Cancer vaccines are one of the important methods of cancer immunotherapy and prevention.
  • the two most important parts of a cancer vaccine are the antigen and the immune adjuvant.
  • the antigen can provide a label for recognition, and the immune adjuvant can effectively enhance the body's immune system to recognize the antigen.
  • Cancer vaccines mainly rely on antigen-specific activation of cancer-specific T cells, that is, cellular immunity.
  • Toll-like receptor agonists are substances that activate innate immune responses and are potential vaccine adjuvants.
  • Toll-like receptors such as Toll-like receptor 3 (TLR3), Toll-like receptor 4, Toll-like receptor 7, Toll-like receptor 8 and Toll-like receptor 9 (TLR9).
  • Poly(I:C) or Poly(ICLC) is an agonist of Toll-like receptor 3, while CpG oligodeoxynucleotide (CpG-ODN) is an agonist of Toll-like receptor 9.
  • Poly(I:C) polyinosinic acid cytidylic acid
  • dsRNA synthetic double-stranded RNA
  • Poly(I:C) can be recognized by TLR3 and induce the activation of NF-kB and the production of cytokines.
  • Poly(ICLC) is a TLR3 agonist of Poly(I:C) that has been appropriately stabilized and modified, and has similar functions to Poly(I:C).
  • CpG-ODN is a synthetic oligodeoxynucleotide (ODN) containing unmethylated cytosine-guanine dinucleotide (CpG), which can simulate bacterial DNA to stimulate a variety of mammalian immune cells, including humans.
  • ODN oligodeoxynucleotide
  • CpG cytosine-guanine dinucleotide
  • Class A CpG-ODN has a palindromic sequence containing CpG dinucleotides as the core, with poly G tails at both ends, and the phosphodiester bond backbone is partially thio-modified.
  • Class B CpG-ODN is a fully thio-modified linear CpG ODN that has strong immunostimulatory activity on B cells, but cannot activate plasmacytoid dendritic cells.
  • Class C CpG-ODN is a type of fully thio-modified CpG ODN that can form dimers through palindromic sequences. It has the activity of both type A and type B CpG-ODN and can activate plasmacytoid dendritic cells. It can also activate B cells.
  • cancer-specific T cells In the process of antigen activation of cancer-specific T cells, in addition to the antigen epitope being presented to the surface of antigen-presenting cells, the assistance of second and third signals is also needed to activate cancer-specific T cells, otherwise the antigen cannot be effectively activated. Cancer-specific T cells.
  • the adjuvant can assist in activating cancer-specific T cells and better exert the function of the vaccine.
  • the current functions of immune adjuvants still need to be further developed in order to fully exert their activation effect on cancer-specific T cells.
  • the present invention provides an immune adjuvant composition, and provides a co-loaded Poly(I:C)/Poly(ICLC) and CpG-ODN (and amino acids, polypeptides, lipids, sugars species, proteins or inorganic salts) and antigenic components of vaccine systems for the prevention or treatment of cancer.
  • the immune adjuvant composition of the present invention can better enhance the efficacy of activating cancer-specific T cell responses and better assist the vaccine to function.
  • the first object of the present invention is to provide an immune adjuvant composition, which is a combination including at least (1) and (2) of the following components: (1) Poly(I:C) Or Poly (ICLC), (2) CpG-ODN, wherein CpG-ODN is at least two of type A CpG-ODN, type B CpG-ODN and type C CpG-ODN, and at least one of them is type B CpG-ODN or C-type CpG-ODN, (3) amino acids, polypeptides, lipids, sugars, proteins or inorganic salts.
  • Class A CpG-ODN includes but is not limited to CpG-ODN 2216, CpG-ODN 1585, CpG-ODN 2336, etc.
  • Class B CpG-ODN includes but is not limited to CpG-ODN 1018, CpG-ODN 2006, CpG-ODN 1826, CpG-ODN 1668, CpG-ODN 2007, CpG-ODN BW006, CpG-ODN SL01, etc.
  • Class C CpG-ODN includes but is not limited to CpG-ODN 2395, CpG-ODN SL03, CpG-ODN M362, etc.
  • the mass ratio of the total mass of CpG-ODN, the mass of Poly(I:C) or Poly(ICLC) and the amino acid, polypeptide, lipid, carbohydrate, protein or inorganic salt is 0.05 -25:1:0.25-50.
  • the amino acid is a positively charged amino acid, preferably a combination of at least two positively charged amino acids, such as: arginine + histidine, arginine + lysine, arginine + lysine + Histidine.
  • the inorganic salt is an inorganic salt that can release H + or acidic substances to form a proton sponge effect, and is preferably ammonium salt, carbonate, bicarbonate, or phosphate.
  • the second object of the present invention is to provide a cancer vaccine loaded with the above immune adjuvant composition.
  • the cancer vaccine includes nanoparticles or microparticles, and antigen components and immune adjuvants loaded on the nanoparticles or microparticles.
  • composition, wherein the antigen component is at least one polypeptide of the same type as the cancer, preferably, a whole cell component antigen derived from cancer cells and/or tumor tissue.
  • the preparation method of the whole cell component antigen includes the following steps: lyse cancer cells or tumor tissues with water or a solution without a dissolving agent, and collect the soluble part as a water-soluble component; dissolve the insoluble part with The soluble part after the agent is dissolved is a non-water-soluble component, and the water-soluble component and the non-water-soluble component are whole cell component antigens; wherein, the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, and deoxycholate , dodecyl sulfate (such as sodium dodecyl sulfate, SDS), glycerol, protein degrading enzyme, albumin, lecithin, 0.1-2000mg/mL inorganic salts, Triton, Tween, DMSO (dimethyl sulfoxide), acetonitrile, ethanol, methanol, DMF (N,N-dimethylformamide), propanol, is
  • cancer cells or tumor tissues are lysed with a dissolving agent, and then the lysate is dissolved to obtain the whole cell component antigen.
  • the water-soluble part and the water-insoluble part of the cell components include the components and components of the entire cell. Among them, unmutated proteins, polypeptides and genes that are the same as normal cell components will not cause an immune response because of the immune tolerance produced during the development of the autoimmune system; while mutations in genes, proteins and polypeptides produced by cancer will not cause an immune response because there is no autoimmune system.
  • the immune tolerance generated during development is therefore immunogenic and can activate the body's immune response against cancer cells.
  • the use of cancer cell-specific immunogenic substances produced by disease mutations in whole cell components can be used for cancer prevention and treatment.
  • the present invention uses a solution containing a dissolving agent to convert components in cells that are insoluble in pure water or aqueous solutions without dissolving agents into soluble in a specific dissolving solution and can be used to prepare cancer vaccines, thereby improving the load of the cancer vaccine. comprehensiveness and immunogenicity of the antigen.
  • the lysis of tumor tissue includes the following steps: cut the tumor tissue into pieces and then grind it, pass it through a cell filter, add an appropriate amount of water or a solution without a dissolving agent, freeze and thaw repeatedly, and may be accompanied by ultrasound to destroy the lysed cells. After the cells are lysed, the lysate is centrifuged, and the supernatant is the water-soluble component; a dissolving agent is added to the precipitate, and the soluble part in the precipitate is the non-water-soluble component.
  • the above is the source of antigen raw materials for preparing cancer vaccines.
  • the lysis of cancer cells includes the following steps: culturing the cancer cell lines, centrifuging, discarding the supernatant, resuspending the cells in pure water or a solution without lytic agent, repeatedly freezing and thawing, and may be accompanied by ultrasound to destroy the lysis.
  • cells after the cells are lysed, centrifuge the lysate, and the supernatant will be the water-soluble component; add a dissolving agent to the precipitate, and the soluble part in the precipitate will be the non-water-soluble component.
  • the above is the antigen raw material for preparing the cancer vaccine. source.
  • the cancer vaccine is connected to targets with active targeting functions, such as mannose, mannan, CD32 antibodies, CD11c antibodies, CD103 antibodies, CD44 antibodies, etc.
  • This target can lead cancer vaccines to target specific tissues or cells, such as dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, and basophils.
  • the antigen component and immune adjuvant are encapsulated inside the nanoparticles or microparticles, and/or loaded on the surface of the nanoparticles or microparticles.
  • Poly(I:C) or Poly(ICLC) and various CpG-ODN can be loaded on the interior and/or surface of nanoparticles or microparticles respectively.
  • the particle size of nanoparticles is 1nm-1000nm; the particle size of micron particles is 1 ⁇ m-1000 ⁇ m; the surface of nanovaccines or micron vaccines prepared using nanoparticles or microparticles is electrically neutral, negatively charged or positively charged.
  • cancer vaccines can be prepared according to the preparation methods developed for nano-sized particles and micron-sized particles, including but not limited to common solvent evaporation methods, dialysis methods, extrusion methods, and hot melt methods.
  • the cancer vaccine is prepared by the double emulsion method in the solvent evaporation method.
  • materials for preparing nanoparticles or microparticles include, but are not limited to, organic synthetic polymer materials, natural polymer materials, or inorganic materials.
  • organic synthetic polymer materials are biocompatible or degradable polymer materials, including but not limited to PLGA (polylactic acid-glycolic acid copolymer), PLA (polylactic acid), PGA (polyglycolic acid), PEG (polyglycolic acid), Ethylene glycol), PCL (polycaprolactone), Poloxamer (Poloxamer), PVA (polyvinyl alcohol), PVP (polyvinylpyrrolidone), PEI (polyethyleneimine), PTMC (polytrimethylene carbonate) ester), polyanhydride, PDON (polydioxanone), PPDO (polydioxanone), PMMA (polymethylmethacrylate), PLGA-PEG, PLA-PEG, PGA-PEG, Polyamino acids,
  • the third object of the present invention is to provide the use of the above immune adjuvant composition or cancer vaccine in the preparation of cancer therapeutic drugs or preventive drugs.
  • the fourth object of the present invention is to provide the use of the above immune adjuvant composition in preparing cellular immune activators.
  • Class B CpG-ODN has strong immunostimulatory activity on B cells, but cannot activate plasmacytoid dendritic cells.
  • Class C CpG-ODN can activate both plasmacytoid dendritic cells and B cells, but Mainly activates B cells. Therefore, theoretically speaking, Class B and Class C CpG-ODN are more effective in activating humoral immunity and less effective in activating cellular immunity; while Class A CpG-ODN can activate plasmacytoid dendritic cells to induce A large amount of type I interferon has weak activity on B cells.
  • Type A should have a better effect on activating cellular immunity but a worse ability to activate humoral immunity.
  • At least one antigen in the cancer vaccine corresponds to the disease treated or prevented by the drug.
  • the present invention at least has the following advantages:
  • Poly(I:C) and different CpG-ODN can activate different targets.
  • Amino acids, polypeptides, lipids, sugars, proteins, inorganic salts, etc. can assist and increase lysosomal escape.
  • the immune adjuvant composition of the present invention can effectively enhance the ability of the loaded adjuvant to assist the antigen in activating specific immune responses, greatly improve the prevention and treatment effect of diseases, and can be used to prepare drugs for the prevention and treatment of cancer.
  • Figure 1 is a schematic diagram of the preparation process and application field of the vaccine when the antigen component of the present invention is a whole cell component; a: a schematic diagram of collecting and preparing nano-vaccines or micro-vaccines from water-soluble components and non-water-soluble components respectively; b : Schematic diagram of using a lysis solution containing a lysing agent to dissolve whole cell components and prepare nano-vaccines or micro-vaccines;
  • Figures 2 to 17 are schematic structural diagrams of nano-sized particles or micron-sized particles loaded with water-soluble components and water-insoluble components.
  • the polypeptides can also be divided into two parts: water-soluble polypeptides and water-insoluble polypeptides. And refer to the loading of whole cell components; among them, 1. Water-soluble components in cells or tissue components; 2. Water-insoluble components in cells or tissue components; 3. Immune adjuvants; 4. Nanoparticles or microparticles ;5. The core part of nanoparticles;
  • Figures 18 to 28 are schematic structural diagrams of nanoparticles or microparticles loaded with water-soluble and non-water-soluble components modified by active targeting targets.
  • the polypeptides can also be divided into water-soluble polypeptides and water-insoluble polypeptides.
  • the two parts of the polypeptide are loaded with reference to the whole cell components; among them, 1. Water-soluble components in cells or tissue components; 2. Water-insoluble components in cells or tissue components; 3. Immune adjuvants; 4. Nanoparticles Or micron particles; 5. The core part in nanoparticles; 6. Targets that can target specific cells or tissues;
  • Figures 29-51 respectively show the co-loading of CpG-ODN and Poly(I:C) or Poly(ICLC) mixed adjuvants with cancer cells or tumor tissue lysates into nano-vaccines or micro-vaccines for prevention in Examples 1-23. Or the experimental results of mouse tumor growth rate and survival time when treating cancer; among them, a. The experimental results of tumor growth rate when nano vaccine or micron vaccine prevents or treats cancer (n ⁇ 8); b.
  • Nano vaccine or micron vaccine prevents or The results of mouse survival experiments when treating other cancers (n ⁇ 8), each data point is the mean ⁇ standard error (mean ⁇ SEM); the significant difference in the tumor growth inhibition experiment in Figure a was analyzed by ANOVA method, Figure The significant difference in b is analyzed by Kaplan-Meier and log-rank test; *** indicates that there is a significant difference compared with the PBS blank control group at p ⁇ 0.0005;## indicates that there is a significant difference compared with the blank nanoparticle + cell lysate control group. If p ⁇ 0.005, there is a significant difference; ### represents p ⁇ 0.0005, which means there is a significant difference compared with the blank nanoparticle + cell lysate control group.
  • & or $ indicates a significant difference at p ⁇ 0.05 compared with the vaccine group loaded with a specific ratio of CpG and Poly(I:C) or polyICLC, or with the vaccine group loaded with only type 1 CpG and Poly(I:C) or polyICLC;&& Or $$ means p ⁇ 0.01, there is a significant difference compared with the vaccine group loaded with a specific ratio of CpG and Poly(I:C) or polyICLC, or with the vaccine group loaded only with type 1 CpG and Poly(I:C) or polyICLC; Indicates that there is a significant difference at p ⁇ 0.01 compared with the vaccine group loaded with CpG and Poly(I:C) or polyICLC mixed adjuvant at a specific ratio and using only one CpG; ⁇ is significantly different from the vaccine group loaded with only the lysate component without loading There is a significant difference at p ⁇ 0.005 compared to the control vaccine group with any immune adjuvant; ⁇ represents a significant difference at p ⁇ 0.05 compared with the
  • the nano-vaccine and/or micro-vaccine system of the present invention can be used to prepare vaccines for preventing and/or treating cancer.
  • the preparation process and application fields are shown in Figure 1.
  • cells or tissues can be lysed and then water-soluble components and water-insoluble components can be collected separately to prepare nano-vaccines or micro-vaccines respectively; or the cells or tissues can be directly lysed using a lysis solution containing a dissolving agent and the whole cells can be dissolved components and prepare nanovaccines or microvaccines.
  • the whole cell components of the present invention can be inactivated or (and) denatured before lysis or (and) after lysis to prepare nano vaccines or micro vaccines, or they can not undergo any inactivation, enzyme treatment or (and) Denaturation treatment directly prepares nano vaccines or micro vaccines.
  • tumor tissue cells undergo inactivation or/and denaturation treatment before lysis.
  • inactivation, enzyme treatment, or/and denaturation treatment may also be performed after cell lysis, or Inactivation, enzyme treatment or (and) denaturation treatment can also be performed before and after cell lysis.
  • the inactivation or denaturation treatment method is ultraviolet irradiation and high-temperature heating. In actual use, radiation can also be used. Inactivation or denaturation treatment methods such as irradiation, high pressure, freeze-drying and formaldehyde.
  • FIG. 2-28 The structural diagram of the vaccine system of the present invention is shown in Figure 2-28.
  • the nanoparticles or microparticles contain immune adjuvants on both the surface and inside; in Figures 6 to 9, the immune adjuvant is only distributed inside the nanoparticles or microparticles; in Figures 10 to 13, the immune adjuvant is only There are no immune-enhancing adjuvants on the outer surface of nanoparticles or microparticles; Figures 14-17 There are no immune-enhancing adjuvants on the inside and outside of nanoparticles or microparticles; water-soluble adjuvants in cells or tissue components in Figures 2, 6, 10 and 14 The water-soluble or non-water-soluble components do not form an obvious core when distributed inside the nanoparticles or microparticles; the distribution of water-soluble or non-water-soluble components in cells or tissue components in Figure 3, Figure 7, Figure 11 and Figure 15 A core part is formed inside the nanoparticles or microparticles, and the core can be generated during the preparation process or formed by using polymers or
  • the cores can be generated during the preparation process or formed by using polymers or inorganic salts;
  • b What is contained inside nanoparticles or microparticles and what is loaded on the surface are non-water-soluble components in cells or tissue components;
  • c nanoparticles or micron
  • the particles are loaded with non-water-soluble components in cells or tissue components, while the water-soluble components in cells or tissue components are loaded on the surface.
  • Nanoparticles or microparticles are loaded with cells or tissue components.
  • the water-soluble components in the components and the water-insoluble components in the cells or tissue components are loaded on the surface;
  • e The water-soluble components and the water-insoluble components in the cells or tissue components are simultaneously contained inside the nanoparticles or microparticles.
  • nanoparticles or microparticles is also loaded with water-soluble components and non-water-soluble components in cells or tissue components; f: water-soluble components in cells or tissue components are simultaneously loaded inside nanoparticles or microparticles and non-water-soluble components, while the surface of nanoparticles or microparticles is only loaded with water-soluble components in cells or tissue components; g: water-soluble components and non-water-soluble components in cells or tissue components are simultaneously loaded inside nanoparticles or microparticles.
  • Nanoparticles or microparticles only carries non-water-soluble components in cells or tissue components
  • h The interior of nanoparticles or microparticles only contains non-water-soluble components in cells or tissue components, while The surface of nanoparticles or microparticles is loaded with both water-soluble and non-water-soluble components in cells or tissue components
  • i The inside of nanoparticles or microparticles only contains water-soluble components in cells or tissue components, while nanoparticles Or the surface of micron particles is loaded with both water-soluble and non-water-soluble components in cells or tissue components
  • the nanoparticles or microparticles contain immune adjuvants on both the surface and inside; in Figures 20 and 21, the immune adjuvant is only distributed inside the nanoparticles or microparticles; in Figures 22 and 23, the nanoparticles or micron particles The particles only contain immune adjuvants on the outer surface; Figure 24- Figure 25 There are no immune adjuvants inside and outside the nanoparticles or microparticles; Figure 26 Cell components and/or immune adjuvants are only distributed inside the nanoparticles or microparticles; Figure 27: Cellular components and/or immune adjuvants are only distributed outside nanoparticles or microparticles; Figure 28: Cellular components and immune adjuvants are distributed inside or outside nanoparticles or microparticles respectively.
  • the non-water-soluble components in the components are loaded on the surface with water-soluble components in the cells or tissue components; d: The water-soluble components contained in the nanoparticles or microparticles are loaded on the surface with the water-soluble components in the cells or tissue components.
  • non-water-soluble components in cells or tissue components are all non-water-soluble components in cells or tissue components; e: water-soluble components and non-water-soluble components in cells or tissue components contained simultaneously inside nanoparticles or microparticles, while the surface of nanoparticles or microparticles It also carries water-soluble components and non-water-soluble components in cells or tissue components at the same time; f: water-soluble components and non-water-soluble components in cells or tissue components are simultaneously loaded inside nanoparticles or microparticles, and nanoparticles Or the surface of micron particles is only loaded with water-soluble components in cells or tissue components; g: Water-soluble components and non-water-soluble components in cells or tissue components are simultaneously contained inside nanoparticles or microparticles, while nanoparticles or micron particles The surface of the particle is only loaded with non-water-soluble components in cells or tissue components; h: The interior of nanoparticles or microparticles is only loaded with non-water-soluble components in cells or tissue components, while the surface of nanop
  • Nanoparticles or microparticles only contain water-soluble components in cells or tissue components, while the surface of nanoparticles or microparticles also loads cells or tissues.
  • Water-soluble and non-water-soluble ingredients in the ingredients In Figures 26-28, the water-soluble or non-water-soluble components in the cells or tissue components loaded by the nanoparticles or microparticles in a, b and c do not form an obvious core when they are distributed inside the nanoparticles or microparticles.
  • the water-soluble or non-water-soluble components in the cells or tissue components loaded by the nanoparticles or microparticles in d, e and f are distributed in a core part inside the nanoparticles or microparticles; the nanoparticles in g, h and i
  • the water-soluble components or non-water-soluble components in the cells or tissue components loaded by the particles or microparticles are distributed in multiple core parts inside the nanoparticles or microparticles; the nanoparticles or microparticles in j, k and l are included
  • the water-soluble or non-water-soluble components in the cells or tissue components are distributed inside the nanoparticles or microparticles to form the outer layer of the core; the nanoparticles or microparticles in a, d, g and j are all loaded with cells or micron particles.
  • nanoparticles and/or microparticles of a specific structure may be used, or one or more nanoparticles and/or microparticles of different structures may be used simultaneously.
  • the specific preparation method of the double emulsion method used in the present invention is as follows:
  • Step 1 Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of medical material.
  • the aqueous solution may contain each component of the cancer cell lysate and an immune adjuvant; each component of the cancer cell lysate is a water-soluble component during preparation or is dissolved in 8M urea or The original non-water-soluble component in dissolving agents such as 6M guanidine hydrochloride.
  • the concentration of the water-soluble components from the cancer cells contained in the aqueous solution or the concentration of the original non-water-soluble components from the cancer cells dissolved in the dissolving agent, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 1ng/mL. , can load enough cancer antigens to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the second predetermined concentration of the medical polymer material ranges from 0.5 mg/mL to 5000 mg/mL, preferably 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 range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is It is 1:1.1-1:5000, preferably 1:10.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., with dichloromethane being preferred;
  • the concentration of the polymer material is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • Step 2 Subject the mixed solution obtained in Step 1 to ultrasonic treatment, stirring, homogenization treatment or 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 to 1500 rpm
  • the stirring time is 0.1 hour to 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment is used to carry out nanometerization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared nanometer and/or micron particles, which 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 ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • This step is for nanonization or micronization.
  • the length of ultrasonic time or stirring speed and time can control the size of the prepared nanoparticles or microparticles. Too long or too short will cause changes in particle size. For this reason, it is necessary to choose the appropriate ultrasound time.
  • 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 50 rpm ⁇ 1500 rpm and the stirring time is 0.5 hours ⁇ 5 hours; during ultrasonic treatment, the ultrasonic power is 50W ⁇ 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.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.
  • the emulsifier aqueous solution is still PVA.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed.
  • Step 5 After centrifuging the mixed solution that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 rpm for greater than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • step 5 when the precipitate obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline), there is no need to freeze-dry, and the subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles can be directly performed.
  • Related experiments may be used directly as nano vaccines or micro vaccines.
  • the precipitate obtained in step 5 needs to be freeze-dried when resuspended in an aqueous solution containing a lyoprotectant, and then freeze-dried before subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles.
  • Trehalose is selected as the freeze-drying protective agent.
  • the fifth predetermined concentration of the freeze-drying protective agent in this step is 4% by mass. The reason why this is set is to not affect the freeze-drying effect during subsequent freeze-drying.
  • Step 6 After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
  • Step 7 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
  • the freeze-dried lyophilized material containing nanoparticles or microparticles and a lyoprotectant is mixed with a seventh predetermined volume of water-soluble components or the original non-water-soluble components dissolved in a dissolving agent such as 8M urea. Get nano vaccines or micro vaccines.
  • 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 volume of the resuspended nanoparticle suspension when the volume of the resuspended nanoparticle suspension is 9 mL, it contains cancer cell lysates or water-soluble components in tumor tissue lysates or original non-water-soluble components dissolved in a dissolving agent.
  • the divided volume is 1mL. In actual use, the volume and proportion of the two can be adjusted as needed.
  • the particle size of nano-vaccines or micro-vaccines is nanoscale or micron-scale, which ensures that the vaccine is engulfed by antigen-presenting cells. In order to improve the phagocytosis efficiency, the particle size must be within an appropriate range.
  • the particle size of the nano vaccine is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 100nm-600nm; the particle size of the micron vaccine is 1 ⁇ m-1000 ⁇ m, more preferably, The particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, and most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • the particle size of the nanoparticle vaccine is 100 nm-600 nm
  • the particle size of the micron vaccine is 1 ⁇ m-5 ⁇ m.
  • the specific method for preparing nano vaccines or micro vaccines using the double emulsion method is as follows:
  • Step 1 Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of medical material.
  • the aqueous phase solution can contain each component of the cancer cell lysate and the immune-enhancing adjuvants poly(I:C) and CpG-ODN, or Poly ICLC and CpG-ODN; the aqueous phase solution contains from The concentration of the water-soluble components of the cancer cells or the concentration of the original non-water-soluble components from the cancer cells dissolved in the dissolving agent, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01ng/mL, which can carry enough cancer Antigens to activate relevant immune responses.
  • the concentration of the immune-enhancing adjuvant in the initial aqueous phase is greater than 0.01ng/mL.
  • the second predetermined concentration of the medical material ranges from 0.5 mg/mL to 5000 mg/mL, preferably 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 range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:1.1-1:5000, preferably 1:10.
  • the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immune adjuvant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., with dichloromethane being preferred;
  • the concentration of the polymer material is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • Step 2 Subject the mixed solution obtained in Step 1 to ultrasonic treatment, stirring, homogenization treatment or 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 to 1500 rpm
  • the stirring time is 0.1 hour to 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.
  • Step 3 Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment, stirring, homogenization treatment or microfluidic treatment.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • This step is for nanonization or micronization.
  • the length of ultrasonic time or stirring speed and time can control the size of the prepared nanoparticles or microparticles. Too long or too short will cause changes in particle size. For this reason, it is necessary to choose the appropriate ultrasound time.
  • 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 50 rpm ⁇ 1500 rpm and the stirring time is 0.5 hours ⁇ 5 hours; during ultrasonic treatment, the ultrasonic power is 50W ⁇ 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.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met, or the subsequent treatment can be performed directly without stirring.
  • the emulsifier aqueous solution is still PVA.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is that 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 a solution containing the water-soluble and/or non-water-soluble components of the whole cell fraction, or the remaining pellet is resuspended in a fifth predetermined volume of a fifth predetermined concentration of the whole cell fraction.
  • Step 6 After centrifuging the mixed solution that meets the predetermined stirring conditions in Step 5 at a rotation speed of greater than 100 RPM for greater than 1 minute, remove the supernatant, and resuspend the remaining sediment in a sixth predetermined volume of solidified liquid.
  • the treatment reagent or mineralization treatment reagent is centrifuged and washed after acting for a certain period of time, and then the seventh predetermined substance containing positively or negatively charged substances is added and acted for a certain period of time.
  • the precipitate obtained in step 6 does not need to be freeze-dried after being resuspended in the seventh predetermined volume of charged substance, and subsequent experiments related to loading cancer cells/tissue lysates on the surface of nanoparticles or microparticles can be directly performed. Or it can be used directly as a nano vaccine or micro vaccine.
  • the precipitate obtained in step 6 is resuspended in an aqueous solution containing a drying protective agent and then subjected to room temperature vacuum drying or freeze vacuum drying. After drying, the subsequent nanoparticles or microparticles surface adsorb cancer cell lysates. Related experiments, or used directly as nano vaccines or micro vaccines.
  • the freeze-drying protective agent is trehalose or a mixed solution of mannitol and sucrose.
  • concentration of the drying protective agent in this step is 4% by mass, which is set so as not to affect the drying effect during subsequent drying.
  • Step 7 After drying the suspension containing the drying protective agent obtained in Step 6, the dried material is used for later use.
  • Step 8 Resuspend an eighth predetermined volume of the nanoparticle-containing suspension obtained in Step 6 in PBS (or physiological saline) or use an eighth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 7
  • PBS or physiological saline
  • the dried material containing nanoparticles or microparticles and a drying protective agent is used directly; or it is used after being mixed with a ninth predetermined volume of water-soluble components or non-water-soluble components.
  • the modification and antigen loading steps of steps 5 to 8 can be repeated multiple times to increase the loading capacity of the antigen.
  • substances with the same charge can be added multiple times or substances with different charges can be added alternately.
  • the volume of the resuspended nanoparticle suspension when the volume of the resuspended nanoparticle suspension is 9 mL, the sum of the volumes of the water-soluble components or the original non-water-soluble components in the cancer cell lysate or tumor tissue lysate is 0.1 -100mL. In actual use, the volume and proportion of the two can be adjusted as needed.
  • Example 1 Adjuvants and melanoma tumor tissue antigens are loaded inside nanoparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nanovaccine co-loaded with immune adjuvants and whole cell components of melanoma tumor tissue, and apply the vaccine to prevent melanoma.
  • B16F10 mouse melanoma cells were used as the cancer model.
  • the B16F10 melanoma tumor tissue was lysed and the water-soluble and non-water-soluble components of the tumor tissue were prepared.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material, and CpG-ODN 1018 (B type), CpG-ODN 2395 (C type) and poly(I:C) were used as immune adjuvants to prepare the loaded nanoparticles using the solvent evaporation method.
  • the nanovaccine and the blank nanoparticles used as a control were prepared by the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used was 7KDa-17KDa.
  • Nanovaccines loaded with water-soluble components and nanoparticles loaded with non-water-soluble components Vaccines are prepared separately and used together.
  • the immune adjuvants used are CpG-ODN 1018, CpG-ODN 2395 and poly(I:C), and the mass ratio between the three is 0.5:1:1.
  • the preparation method is as mentioned above.
  • PLGA nanoparticles encapsulate immune adjuvants and whole cell components in the nanovaccine.
  • the average particle size of the nanovaccine loaded with whole cell components is about 280nm, and the surface potential of the nanovaccine is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components, and the total mass of adjuvants used per 1 mg of PLGA nanoparticles is 0.05 mg, of which CpG-ODN 1018 is 0.01mg, CpG-ODN 2395 is 0.02mg, and poly(I:C) is 0.02mg.
  • the particle size of the blank nanoparticles loaded with only 0.05mg of immune adjuvant (CpG-ODN 1018 is 0.01mg, CpG-ODN 2395 is 0.02mg, poly(I:C) is 0.02mg) is about 270nm, and the blank nanoparticles are prepared separately.
  • the water-soluble components from B16F10 tumor tissue and the original non-water-soluble components dissolved in 8M urea are used as raw materials to prepare a vaccine that only loads the whole cell components of B16F10 tumor tissue.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide. Components, the surface potential of nano-vaccine is about -5mV.
  • the particle size of the control nanovaccine is 280nm, and each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components.
  • the total mass of adjuvants used per 1 mg of PLGA nanoparticles is 0.05 mg, of which CpG-ODN 1018 is 0.0125 mg and CpG-ODN 2395 is 0.0125mg, poly(I:C) is 0.025mg, and the surface potential of the nanovaccine is about -5mV.
  • the control groups in this study were the PBS group and the blank nanoparticles + free tissue lysate group.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice.
  • the dosing schedule of the nanovaccine group is as follows: 100 ⁇ L of 1 mg PLGA nanovaccine loaded with water-soluble components and 100 ⁇ L of loaded original substance were subcutaneously injected on the 42nd day, the 35th day, the 28th day, the 21st day, and the 7th day before melanoma vaccination. 1 mg PLGA nanovaccine with non-water-soluble components; 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse on day 0.
  • the plan for the PBS control group is as follows: subcutaneously inject 200 ⁇ L of PBS on the 42nd, 35th, 28th, 21st, and 7th days before melanoma inoculation; on the 0th day, subcutaneously inject 200 ⁇ L of PBS into the lower right corner of the back of each mouse. Inoculate 1.5 ⁇ 10 B16F10 cells.
  • Blank nanoparticles + free lysate control group 200 ⁇ L of blank nanoparticles and an equal amount of free lysate were subcutaneously injected on days 42, 35, 28, 21, and 7 days before melanoma inoculation; blank nanoparticles and Free lysates were injected at different sites; each mouse was subcutaneously inoculated with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back on day 0.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew and grew rapidly.
  • the efficacy of the nanovaccine group was better than that of the PBS control group and the blank nanoparticle control group, and the nanovaccine loaded with a 1.5:1 (CpG-ODN:poly(I:C)) ratio mixed adjuvant and tumor tissue whole cell components was treated Most of the tumors in the mice in the group disappeared after vaccination, which was better than the vaccine loaded with a 1:1 (CpG-ODN:poly(I:C)) ratio of mixed adjuvant and tumor tissue whole cell components.
  • the nanovaccine of the present invention loaded with specific proportions of multiple CpG-ODN and Poly(I:C) and antigen components has a good preventive effect on melanoma.
  • Example 2 Adjuvants and polypeptides loaded inside nanoparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nanovaccine co-loaded with an immune adjuvant and a polypeptide, and apply the vaccine to prevent melanoma.
  • B16F10 mouse melanoma cells were used as the cancer model.
  • the organic polymer material PLGA is used as the nanoparticle skeleton material
  • poly(I:C) and CpG-ODN 2006 (B type) and CpG-ODN 2216 (A type) are used as immune adjuvants and the loaded adjuvant and Nanovaccines based on peptide antigens.
  • the nanovaccine was then used to prevent melanoma.
  • This embodiment uses water-soluble polypeptide antigen B16-M20 (TUBB3, FRRKAFLHWYTGEMDEMEMEMEAESNM), B16-M24 (DAG1, TavitppttttkarvstpkPatpstd), B16-M27 (ReGVELCPGNKYEMRRHGTTTTTTTTTTT) HSLVIHD) and 8m urea water solution (including 500 mm sodium chloride) dissolved in water insoluble polypeptide antigen B16 -M05(Eef2,FVVKAYLPVNESFAFTADLRSNTGGQA),B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ), and TRP2:180-188(SVYDFFVWL).
  • 8M urea aqueous solution containing 500mM sodium chloride
  • the nanovaccine and the control blank nanoparticles and nanoparticles loaded with multiple polypeptides were prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used was 24KDa-38KDa.
  • the adjuvants are CpG-ODN 2006 (Class B), CpG-ODN 2216 (Class A) and poly(I:C), and the mass ratio of the three is 1:1:0.5.
  • the preparation method is as described above.
  • the average particle size of the nanovaccine loaded with peptides is about 270nm, and the surface potential of the nanovaccine is about -5mV; every 1 mg of PLGA nanoparticles is loaded with approximately 70 ⁇ g of polypeptide components, and a total of 0.05 mg of immune adjuvant is used inside and outside the 1 mg of PLGA nanoparticles, of which CpG -ODN 2006 is 0.02mg, CpG-ODN 2216 is 0.02mg, poly(I:C) is 0.01mg.
  • the particle size of blank nanoparticles containing only immune adjuvant is about 250 nm.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 70 ⁇ g of peptide components.
  • the immune adjuvants used inside and outside each 1 mg of PLGA nanoparticles are 0.05 mg in total, including 0.025 mg for CpG-ODN 2006 and 0.025 mg for CpG-ODN 2216. is 0.024mg, poly(I:C) is 0.001mg.
  • the control groups in this study were the PBS group and the blank nanoparticles + free peptide group.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice.
  • the dosing schedule of the nanovaccine group is as follows: 200 ⁇ L of 2mg PLGA nanovaccine was injected subcutaneously on days 49, 42, 35, 28 and 14 before melanoma vaccination; on day 0, each mouse was given The mice were inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back.
  • the plan for the PBS control group is as follows: subcutaneously inject 200 ⁇ L PBS on days 49, 42, 35, 28, and 14 days before inoculation of melanoma; on day 0, subcutaneously inoculate 1.5 ⁇ 10 5 in the lower right corner of the back of each mouse. B16F10 cells.
  • Blank nanoparticles + free peptide control group 200 ⁇ L of blank nanoparticles and an equal amount of free lysate loaded with the vaccine were subcutaneously injected on days 49, 42, 35, 28 and 14 days before melanoma inoculation; blank nanoparticles Granules and free peptides were injected at different sites; on day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse.
  • the tumors of mice in both the PBS control group and the blank nanoparticle control group grew and grew rapidly.
  • the efficacy of the nanovaccine group was better than that of the PBS control group and the blank nanoparticle control group, and the mice in the nanovaccine treatment group loaded with a 4:1 (CpG-ODN:poly(I:C)) ratio mixed adjuvant and polypeptide antigen Most of the tumors disappeared after vaccination, which was better than the vaccine loaded with 49:1 (CpG-ODN:poly(I:C)) ratio of mixed adjuvant and polypeptide antigen.
  • the nanovaccine of the present invention loaded with specific proportions of multiple CpG and Poly(I:C) and antigen components has a good preventive effect on melanoma.
  • Example 3 Adjuvants and melanoma cancer cells are loaded inside nanoparticles for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nanovaccine co-loaded with an immune adjuvant and whole cell components of melanoma cancer cells, and use the vaccine to treat melanoma.
  • B16F10 mouse melanoma cells were used as the cancer model.
  • B16F10 melanoma cancer cells were first lysed and water-soluble and water-insoluble fractions were prepared.
  • the organic polymer material PLGA is used as the nanoparticle skeleton material, and poly(I:C), CpG-ODN 1018 (B type), CpG-ODN 1826 (B type) and CpG-ODN 2336 (A type) are used as immune
  • the adjuvant uses a solvent evaporation method to prepare nano vaccines loaded with water-soluble components and non-water-soluble components of cancer cells. The nanovaccine is then used to treat melanoma.
  • the cultured B16F10 cancer cell line was removed from the culture medium, centrifuged at 400g for 5 minutes, resuspended in PBS and washed twice, then resuspended the cancer cells in ultrapure water, and repeatedly frozen and thawed five times. Ultrasound was used to assist during the freezing and thawing process.
  • the cancer cells were lysed, and then centrifuged at 10,000 g for 5 minutes, and the supernatant was collected as the water-soluble fraction. Dissolve the precipitated part with 8M urea, which is the dissolved original non-water-soluble component. Mixing the water-soluble components from B16F10 cancer cells and the original non-water-soluble components dissolved in 8M urea at a mass ratio of 1:1 is the source of raw materials for vaccine preparation.
  • the nano vaccine is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nano particle preparation material PLGA used is 7KDa-17 KDa.
  • the adjuvant and antigen components are contained in the nano vaccine.
  • the immune adjuvant used For poly(I:C), CpG-ODN 1018, CpG-ODN 1826 and CpG-ODN 2336. The preparation method is as described above.
  • the average particle size of the nanovaccine loaded with whole cell components is about 320nm; the surface potential of the nanovaccine is about -5mV; each 1mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein or peptide components, and the adjuvant used per 1mg PLGA nanoparticle is 0.05mg.
  • poly(I:C) is 0.01mg
  • CpG-ODN 1018 is 0.01mg
  • CpG-ODN 1826 is 0.01mg
  • CpG-ODN 2336 is 0.02mg.
  • the preparation method of the control nano-vaccine is the same, with a particle size of 320 nm and a vaccine surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components; each 1 mg of PLGA nanoparticles uses an adjuvant of 0.05 mg, poly(I: C) is 0.049mg, CpG-ODN 1018 is 0.0003mg, CpG-ODN 1826 is 0.0003mg, and CpG-ODN 2336 is 0.0004mg.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice.
  • the dosing schedule of the nanovaccine group is as follows: 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of each mouse’s back on day 0; on days 4, 7, 10, and 15 after inoculation with melanoma and 100 ⁇ L of 2 mg PLGA nanovaccine were subcutaneously injected on the 20th day.
  • the plan for the PBS control group is as follows: 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse on day 0; on the 4th, 7th, 10th, 15th, and On the 20th day, 200 ⁇ L PBS was injected subcutaneously.
  • the tumors of mice in the PBS control group all grew and grew rapidly.
  • the efficacy of the nanovaccine group was better than that of the PBS control group, and the ratio of loading 0.5:0.5:1:0.5 (CpG-ODN 1018: CpG-ODN 1826: CpG-ODN 2336: poly(I:C)) mixed adjuvant and tumor
  • the tumors of the mice in the nanovaccine treatment group of tissue whole cell components mostly disappeared after vaccination, which was better than the load 3:3:4:490 (CpG-ODN 1018: CpG-ODN 1826: CpG-ODN 2336: poly(I :C))
  • a vaccine that mixes adjuvant and tumor tissue whole cell components in a proportion.
  • the nanovaccine of the present invention loaded with a specific proportion of multiple CpG and Poly(I:C) and antigen components has a good therapeutic effect on melanoma.
  • Example 4 Adjuvants and melanoma tumor tissue are loaded inside nanoparticles for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nanovaccine co-loaded with immune adjuvants and whole cell components of melanoma tumor tissue, and use the vaccine to treat melanoma.
  • B16F10 mouse melanoma cells were used as the cancer model.
  • B16F10 melanoma tumor tissue was first lysed and water-soluble and non-water-soluble fractions were prepared.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material, and poly(I:C), CpG-ODN 2336 (Class A) and CpG-ODN 1018 (Class B) were used as immune adjuvants to prepare loaded nanoparticles using the solvent evaporation method.
  • Nanovaccines of water-soluble and non-water-soluble components of cancer cells The nanovaccine is then used to treat melanoma.
  • Example 2 Same as Example 1. Mixing the water-soluble components and the original non-water-soluble components dissolved in 5% sodium deoxycholate at a mass ratio of 1:1 is the raw material source for vaccine preparation.
  • the nano vaccine is prepared by the double emulsion method.
  • the molecular weight of the nano particle preparation material PLGA used is 24KDa-38 KDa.
  • the adjuvant and antigen components are contained in the nano vaccine.
  • the immune adjuvant used is poly(I: C), CpG-ODN 2336 and CpG-ODN 1018.
  • the preparation method is as described above.
  • the average particle size of the nanovaccine loaded with whole cell components is about 350nm; the surface potential of the nanovaccine is about -5mV; each 1mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein or peptide components, and the adjuvant used per 1mg PLGA nanoparticle is 0.03mg.
  • poly(I:C) is 0.01mg
  • CpG-ODN 2336 is 0.01mg
  • CpG-ODN 1018 is 0.01mg.
  • the preparation method of the control nanovaccine 1 is the same, with a particle size of 340nm and a vaccine surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components; each 1 mg of PLGA nanoparticles uses 0.03 mg of adjuvant, of which 0.01 mg poly(I:C), 0.01mg CpG-ODN 2336 and 0.01mg CpG-ODN 2216.
  • the preparation method of the control nanovaccine 2 is the same, with a particle size of 340nm and a vaccine surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components; each 1 mg of PLGA nanoparticles uses 0.03 mg of adjuvant, of which 0.01 mg CpG-ODN 1018, 0.01mg CpG-ODN 2336 and 0.01mg CpG-ODN 2216;
  • the nanovaccine of the present invention loaded with a specific proportion of multiple CpG and Poly(I:C)
  • Example 5 Water-soluble cell components of melanoma and lung cancer cells are loaded inside and on the surface of microparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a micron vaccine loaded only with the water-soluble part of melanoma and lung cancer cell components, and use the vaccine to prevent melanoma.
  • B16F10 melanoma and LLC lung cancer cells were first lysed to prepare water-soluble components and non-water-soluble components. Then, the organic polymer material PLGA (24KDa-38KDa) is used as the micron particle skeleton material, and poly(I:C) and two CpG-ODN are used as immune adjuvants to prepare micron vaccines loaded with water-soluble components of whole cells. And apply this micron vaccine to prevent melanoma.
  • B16F10 cells or LLC cells Collect a certain amount of B16F10 cells or LLC cells, remove the culture medium and freeze them at -20°C. Add a certain amount of ultrapure water and process the cells by heating and ultraviolet irradiation. Then freeze and thaw repeatedly for more than 3 times, accompanied by ultrasonic damage to lyse the cells. . After the cells are lysed, centrifuge the lysate at 8000g for 5 minutes and take the supernatant, which is the water-soluble component of B16F10 melanoma or LLC lung cancer cells that is soluble in pure water. The water-soluble components derived from the two cancer cell lysates obtained above are mixed at a mass ratio of 1:1 to form the antigen source for preparing micron vaccines.
  • the micron vaccine and the blank microparticles used as controls were prepared using the double emulsion method in the solvent evaporation method.
  • the micron particle preparation material used was PLGA, and the immune adjuvants used were poly(IC) and CpG-ODN 2006. (Class B) and CpG-ODN2216 (Class A), the mass ratio of the three is 2:2:1.
  • the antigen component is contained inside the micron vaccine and adsorbed on the surface of the micron particles, while the adjuvant is only contained inside the micron vaccine.
  • the preparation method is as described above.
  • the particle size of the micron vaccine obtained after loading cell components and immune adjuvants on the surface of the micron particles is 2.10 ⁇ m, and the surface potential is about -5mV.
  • Each 1 mg of PLGA micron particles is loaded with 150 ⁇ g of protein or peptide components, and 0.05 mg of adjuvant is used, in which Poly (I:C)0.02mg, CpG-ODN2006 0.02mg, CpG-ODN2216 0.01mg.
  • Control Micron Vaccine 1 only uses one kind of CpG and poly(I:C) as a mixed adjuvant, with a particle size of 2.10 ⁇ m and a surface potential of -5mV.
  • Each 1 mg PLGA micron particle is loaded with 150 ⁇ g of protein or peptide components, and an adjuvant of 0.05 is used. mg, including Poly(IC)0.02mg and CpG-ODN2006 0.03mg.
  • the particle size of the control micron vaccine 2 is 2.10 ⁇ m, the surface potential is -5mV, each 1mg PLGA micron particle is loaded with 150 ⁇ g protein or peptide component, and an adjuvant of 0.05mg is used, including Poly(IC) 0.025mg, CpG-ODN 2006 0.02mg, CpG-ODN 2216 0.005mg.
  • Melanoma tumor-bearing mice were prepared from 6-8 week old female C57BL/6 mice.
  • the protocol for the micron vaccine group is as follows: 200 ⁇ L of 2 mg PLGA micron vaccine loaded with water-soluble components in cancer cell lysate was subcutaneously injected on the 35th, 28th, 21st, and 14th days before melanoma inoculation; Each mouse was inoculated subcutaneously with 1.5 ⁇ 10 5 B16F10 cells on the lower right side of the back.
  • the plan for the PBS blank control group is as follows: subcutaneously inject 200 ⁇ L PBS on the 35th, 28th, 21st, and 14th days before inoculation of melanoma; on the 0th day, subcutaneously inoculate 1.5 ⁇ 10 5 B16F10 cells.
  • the tumor volume growth rate of mice in the micron vaccine administration group was significantly slower and the survival period of mice was significantly prolonged.
  • Example 6 Liver cancer tumor tissue lysis components loaded inside and on the surface of micron particles for the prevention of liver cancer
  • a micron vaccine loaded with liver cancer tumor tissue lysate components is prepared, and the vaccine is used to prevent liver cancer.
  • the mouse liver cancer tumor tissue lysate components are loaded inside and on the surface of micron particles to prepare micron vaccines.
  • mouse liver cancer tumor tissue was obtained, and 8M urea was used to lyse the tumor tissue and dissolve the tumor tissue lysate components.
  • PLGA 38KDa-54KDa
  • Poly ICLC Poly ICLC
  • CpG-ODN M362 Category C
  • CpG-ODN2216 A class
  • Each C57BL/6 mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 cells or 2 ⁇ 10 6 LLC lung cancer cells under the armpit, and each mouse was sacrificed when the inoculated tumor grew to a volume of approximately 1000 mm 3 . mice and remove tumor tissue. The tumor tissue was minced and passed through a cell mesh, and then lysed with 8M urea and dissolved.
  • the double emulsion method in the solvent evaporation method was used to prepare micron vaccines and blank micron particles as controls.
  • the micron particle preparation material used was PLGA, and the immune adjuvants used were Poly ICLC, CpG-ODN M362 and CpG. -ODN 2216, the mass ratio of the three is 1:3:1.
  • the antigen component is contained inside the micron vaccine and adsorbed on the surface of the micron particles, while the adjuvant is only contained inside the micron vaccine.
  • the antigen components and adjuvants are encapsulated inside the microparticles using the dissolution and evaporation method, then centrifuged at 8000g for 15 minutes, the precipitate is collected and 100 mg of PLGA microparticles are resuspended in 4% trehalose solution, then freeze-dried for 48 hours and refrigerated. spare.
  • 10 mg PLGA micron particles are resuspended in 0.9 mL PBS or normal saline, and then mixed with 0.1 mL of cell lysate (60 mg/mL) containing 8 M urea and immune adjuvant (2 mg/mL, with a mass ratio of Poly
  • the resulting micron vaccine has a particle size of 2.10 ⁇ m and a surface potential of about -5mV.
  • Each 1 mg PLGA micron particle is loaded with 150 ⁇ g of protein or peptide components.
  • An adjuvant of 0.05 mg is used, including Poly ICLC 0.01 mg, CpG-ODN M362 0.03 mg, and CpG- ODN 2216 0.01mg.
  • the particle size of the control micron vaccine is 2.10 ⁇ m, the surface potential is -5mV, each 1mg PLGA micron particle is loaded with 150 ⁇ g protein or peptide components, and Poly ICLC, CpG-ODN M362 and CpG-ODN 2216 are used as adjuvants, of which Poly ICLC 0.025mg , CpG-ODN M362 0.0125mg, CpG-ODN 2216 0.0125mg.
  • the particle size of the blank microparticles loaded only with immune adjuvants is 2.00 ⁇ m.
  • 8M urea containing equal amounts of Poly ICLC, CpG-ODN M362 and CpG-ODN 2216 was used to replace the corresponding lysate components.
  • Hepa 1-6 liver cancer tumor-bearing mice were prepared from 6-8 week old female C57BL/6.
  • the protocol for the PBS blank control group is as follows: 200 ⁇ L PBS was injected subcutaneously on the 49th, 42nd, 35th, 28th and 14th days before inoculation of liver cancer cells. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit.
  • Blank microparticles + free lysate control group subcutaneously inject 200 ⁇ L of blank microparticles and the same amount of the vaccine on the 49th, 42nd, 35th, 28th and 14th days before inoculation of liver cancer cells. Free lysate; blank microparticles and free cell lysate were injected at different sites. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit. In the experiment, the mouse tumor growth monitoring method was the same as above.
  • Example 7 Lung cancer and liver cancer tumor tissue whole cell components loaded inside nanoparticles for the prevention of liver cancer
  • This example uses mouse liver cancer as a cancer model to illustrate how to prepare nano vaccines loaded with whole cell components of lung cancer and liver cancer tumor tissues, and apply the vaccine to prevent liver cancer.
  • lung cancer and liver cancer tumor tissues are lysed to prepare water-soluble and non-water-soluble components of the whole cell component, and the water-soluble components are mixed in a mass ratio of 1:1, and the non-water-soluble components are also mixed in a mass ratio of 1:1. Mix in a ratio of 1:1.
  • PLGA was used as the nanoparticle skeleton material
  • Poly(I:C), CpG-ODN 1826 (B type) and CpG-ODN 1018 (B type) were mixed in a mass ratio of 1:1:1 as an immune adjuvant to prepare the load.
  • Nano-vaccines containing water-soluble components or loaded with non-water-soluble components are used to prevent liver cancer by mixing nano-vaccines loaded with water-soluble components and nano-vaccines loaded with non-water-soluble components.
  • 1.0 ⁇ 10 6 LLC cells or 1.0 ⁇ 10 6 Hepa 1-6 cells were subcutaneously inoculated on the back of each C57BL/6 mouse.
  • the mice were sacrificed and the tumor tissue was removed.
  • the tumor tissue was cut into pieces and then ground.
  • An appropriate amount of pure water was added through a cell filter and frozen and thawed 5 times repeatedly, accompanied by ultrasound to destroy the lysed tissue cells.
  • the nano-vaccine is prepared using the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa.
  • Poly(I:C), CpG-ODN 1826 and CpG-ODN 1018 are calculated by mass. Use as a mixed adjuvant in a ratio of 1:1:1.
  • a mixture of water-soluble components and a mixture of non-water-soluble components are prepared separately into nano vaccines and then used together. The antigen components and adjuvants are only loaded into the nano vaccines in the form of nanoparticles.
  • the preparation method is the same as above.
  • the particle size of the nanovaccine is about 280nm, and the surface potential is about -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, and a total of 0.03 mg of adjuvant is used for each 1 mg of PLGA nanoparticles.
  • the particle size of the blank nanoparticles is about 250 nm. When preparing the blank nanoparticles, solutions containing equal amounts of adjuvants are used to replace the corresponding water-soluble components and non-water-soluble components.
  • the particle size of the control nanovaccine is about 280nm, and the surface potential is about -6mV.
  • Each 1mg of PLGA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components.
  • Each 1mg of PLGA nanoparticles uses a total of 0.03mg of adjuvant Poly(I:C) or CpG- ODN 1826+CpG-ODN 1018 total 0.03mg
  • Hepa 1-6 liver cancer tumor-bearing mice were prepared from 6-8 week old female C57BL/6 mice. On days 49, 42, 35, 28 and 14 before inoculation of liver cancer cells, 100 ⁇ L of 1 mg PLGA water-soluble component nanovaccine and 100 ⁇ L of 1 mg PLGA non-water-soluble component nanovaccine were subcutaneously injected, respectively. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit.
  • the protocol for the PBS blank control group is as follows: 200 ⁇ L PBS was injected subcutaneously on the 49th, 42nd, 35th, 28th and 14th days before inoculation of liver cancer cells.
  • each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit.
  • Blank nanoparticles + free lysate control group subcutaneously inject 200 ⁇ L of blank nanoparticles and the same amount of the vaccine on the 49th, 42nd, 35th, 28th and 14th days before inoculation of liver cancer cells. Free lysate; blank nanoparticles and free cell lysate were injected at different sites.
  • each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit. In the experiment, the mouse tumor growth monitoring method was the same as above.
  • Example 8 Pancreatic cancer tumor tissue lysis components are loaded inside nanoparticles for the treatment of pancreatic cancer
  • mouse pancreatic cancer as a cancer model to illustrate how to prepare a nanovaccine loaded with pancreatic cancer tumor tissue lysate components and use the vaccine to treat pancreatic cancer.
  • mouse pancreatic cancer tumor tissue was obtained and lysed to prepare water-soluble fractions and original non-water-soluble fractions dissolved in 6M guanidine hydrochloride.
  • Nanovaccines were prepared using PLGA (molecular weight 7KDa-17KDa) as the nanoparticle skeleton material, and Poly(I:C), CpG-ODN 2395 and CpG-ODN 2216 as immune adjuvants.
  • Pan02 pancreatic cancer cells were subcutaneously inoculated into the armpit of each C57BL/6 mouse. When the inoculated tumors in each mouse grew to a volume of approximately 1000 mm 3 , the mice were sacrificed and the tumor tissues were harvested.
  • the lysis method of tumor tissue and the collection method of each component are the same as above, except that 6M guanidine hydrochloride is used instead of 8M urea.
  • the double emulsion method in the solvent evaporation method is used to prepare the nano-vaccine.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa.
  • the ratio of 1:0.6:0.6 is used as a mixed adjuvant.
  • a mixture of water-soluble components and a mixture of non-water-soluble components are prepared separately into nano vaccines and then used together.
  • the antigen components and adjuvants are only loaded into the nano vaccines in the form of nanoparticles.
  • the preparation method is the same as above.
  • the particle size of the nanovaccine is about 270nm, and the surface potential is about -5mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components.
  • a total of 0.044 mg of adjuvant is used for each 1 mg of PLGA nanoparticles, of which Poly(I:C) is 0.02 mg, CpG-ODN 2395 is 0.012mg: CpG-ODN 2216 is 0.012mg.
  • the control nanovaccine has a particle size of about 280nm and a surface potential of about -5mV.
  • Each 1mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components.
  • Each 1mg of PLGA nanoparticles uses a total of 0.044mg of adjuvants, of which Poly(I:C) is 0.02mg, CpG-ODN 1585 is 0.012mg: CpG-ODN 2216 is 0.012mg.
  • the particle size of the control nanovaccine 2 is about 280nm, the surface potential is about -6mV, each 1mg PLGA nanoparticle is loaded with about 80 ⁇ g protein or peptide components, and each 1mg PLGA nanoparticle uses a total of 0.044mg of adjuvant, of which CpG1585 is 0.02mg, CpG -ODN 2395 is 0.012mg: CpG-ODN 2216 is 0.012mg.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected to prepare pancreatic cancer mice. On day 0, 1 ⁇ 10 6 Pan02 cells were subcutaneously inoculated into the lower right corner of the back of each mouse.
  • the vaccine group was subcutaneously injected with 100 ⁇ L of 1 mg PLGA nanovaccine loaded with water-soluble components and 100 ⁇ L of 1 mg PLGA nanovaccine loaded with non-water-soluble components on the 4th, 7th, 10th, 15th and 20th days respectively.
  • the PBS blank control group was injected subcutaneously with 200 ⁇ L PBS on the 4th, 7th, 10th, 15th and 20th days respectively.
  • the mouse tumor monitoring and volume calculation methods were the same as above.
  • Example 9 Whole cell components loaded inside mannose-modified microparticles for the prevention of breast cancer
  • This example uses mouse breast cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with whole cell components of breast cancer tumor tissue and cancer cells, and apply the vaccine to prevent breast cancer.
  • particle size, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • mouse breast cancer and lung cancer cancer cells were first obtained and lysed to prepare water-soluble components and original non-water-soluble components dissolved in 8M urea. Then, PLGA and mannose-modified PLGA were used as micron particle framework materials, and Poly(I:C), CpG-ODN 1018 (B type) and CpG-ODN 1826 (B type) were used as immune adjuvants to prepare by solvent evaporation method.
  • Micron vaccine The micron vaccine has the ability to target dendritic cells.
  • the micron vaccine and the empty micron particles used as controls were prepared by the double emulsion method.
  • the molecular weight of the microparticle preparation material PLGA (50:50) is 24KDa-38KDa
  • the molecular weight of the mannose-modified PLGA (50:50) used is 24KDa-38KDa.
  • the mass ratio of unmodified PLGA and mannose-modified PLGA was 8:2.
  • the preparation method is as described above, and the antigen component and adjuvant are co-loaded in the micron vaccine.
  • the preparation method is as described above.
  • the average particle size of the micron vaccine is about 1.50 ⁇ m
  • the average surface potential is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA micron vaccine uses 0.05 mg of adjuvant, of which CpG-ODN 1018 is 0.025 mg. , CpG-ODN 1826 is 0.023mg, Poly(I:C) is 0.002mg.
  • the particle size of the blank microparticles is about 1.45 ⁇ m. When the blank microparticles are prepared, they are loaded with the same amount of adjuvant but not with the antigen component.
  • the average particle size of the control micron vaccine is about 1.50 ⁇ m, and the average surface potential is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA micron vaccine uses 0.05 mg of adjuvant, of which CpG-ODN 1018 is 0.025. mg, CpG-ODN 1826 is 0.024mg, Poly(I:C) is 0.001mg.
  • Example 10 Melanoma tumor tissue and whole cell components of cancer cells are loaded into nano vaccines for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model and Poly(I:C), CpG-ODN 1018 and CpG-ODN 2006 as immune adjuvants to illustrate how to prepare whole cell components loaded with melanoma tumor tissue and cancer cells. Nano-vaccine and its application in the treatment of melanoma.
  • the water-soluble components and non-water-soluble components of liver cancer and melanoma tumor tissues are first lysed and mixed at a ratio of 3:1 respectively. Then, nanovaccines were prepared using PLGA as the nanoparticle skeleton material using a solvent evaporation method.
  • the lysis of tumor tissue and cancer cells and the collection of lysates are the same as above.
  • Water-insoluble components were dissolved using 5% SDS.
  • the mixture of water-soluble components and the mixture of non-water-soluble components are mixed in a mass ratio of 2:1, which is the antigen component for preparing nano vaccines.
  • the nanovaccine prepared in this example and the blank nanoparticles used as controls were prepared by the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLGA (50:50) is 24KDa-38KDa
  • the molecular weight of the mannose-modified PLGA (50:50) used is 24KDa-38KDa
  • the mass ratio of unmodified PLGA and mannose-modified PLGA is 9: 1.
  • the preparation method is as described above, and the antigen component and adjuvant are co-loaded into the nanovaccine.
  • the average particle size of the nanovaccine is about 280nm, and the average surface potential is about -4mV.
  • Each 1mg of PLGA nanovaccine is loaded with 95 ⁇ g of protein or peptide components.
  • Each 1mg of PLGA nanovaccine uses 0.05mg of adjuvant, of which CpG-ODN 1018 is 0.025mg.
  • CpG-ODN 2006 is 0.02mg
  • Poly(I:C) is 0.005mg.
  • the average particle size of the control nanovaccine 1 is about 280nm, the average surface potential is about -5mV, each 1mg PLGA nanovaccine is loaded with 95 ⁇ g protein or peptide components, and each 1mg PLGA nanovaccine uses 0.05mg adjuvant, of which CpG-ODN 1018 is 0.025 mg, CpG-ODN 2006 is 0.024mg, Poly(I:C) is 0.001mg (mass ratio is 25:24:1).
  • the average particle size of the control nanovaccine 2 is about 280nm, the average surface potential is about -5mV, each 1mg PLGA nanovaccine is loaded with 95 ⁇ g protein or peptide components, and each 1mg PLGA nanovaccine uses 0.05mg adjuvant, of which CpG-ODN 1018 is 0.02 mg, CpG-ODN 2006 is 0.005mg, Poly(I:C) is 0.025mg (mass ratio is 4:1:5).
  • mice Female C57BL/6 mice were selected as model mice to prepare tumor-bearing mice. Each group was subcutaneously inoculated with 1.5 ⁇ 10 5 B16F10 melanoma cells on the lower right side of the back of each mouse on day 0.
  • the vaccine group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanovaccine on the 4th, 7th, 10th, 15th and 20th days after tumor inoculation.
  • the PBS blank control group was injected subcutaneously with 200 ⁇ L PBS on the 4th, 7th, 10th, 15th and 20th days after tumor inoculation.
  • the method for monitoring mouse tumor growth is the same as above.
  • the tumor growth rate and mouse survival time in the vaccine treatment group were significantly different.
  • the tumors in mice in the vaccine group partially disappeared after vaccination.
  • the preventive effect of the nanovaccine with total CpG-ODN and Poly(I:C) mass ratio of 9:1 as adjuvant is better than that of total CpG-ODN with Poly(I:C) mass ratio of 49:1 and 1:1 as adjuvant. dose of nanovaccine.
  • Example 11 8M urea dissolves breast cancer tumor tissue components and loads them into nanoparticles to treat breast cancer
  • This example illustrates how to use 8M urea to dissolve whole cell components and prepare nanovaccines loaded with whole cell components to treat breast cancer.
  • 4T1 mouse triple-negative breast cancer was used as a cancer model.
  • the breast cancer tumor tissue cells were inactivated and denatured, and the tumor tissue was lysed with 8M urea and the whole cell components were dissolved.
  • PLGA as the nanoparticle skeleton material
  • Poly(I:C) Poly(I:C)
  • CpG-ODN 1018 (B type) and CpG-ODN M362 (C type) as immune adjuvants
  • a solvent evaporation method was used to prepare tumor tissue-loaded whole cells. Components of nanovaccines.
  • 4 ⁇ 10 5 4T1 cells were subcutaneously inoculated into the right armpit of BALB/c mice.
  • the mice were sacrificed and the tumor tissues were removed. Cut the tumor tissue into pieces, add collagenase and incubate at 37°C for 15 minutes, then grind and filter through a cell filter to collect the filtered tumor tissue cells.
  • the obtained tumor tissue cells were inactivated and denatured using ultraviolet light and high-temperature heating respectively, and then an appropriate amount of 8M urea was used to lyse the breast cancer tumor tissue cells and dissolve the lysate, which is the source of the antigen component for vaccine preparation.
  • the nanovaccine prepared in this example and the blank nanoparticles used as controls were prepared by the double emulsion method.
  • the molecular weight of nanoparticle preparation material PLGA (50:50) is 7KDa-17KDa.
  • the preparation method is as described above, and the antigen component and adjuvant are co-loaded into the nanovaccine.
  • the average particle size of the nanovaccine is about 250nm, and the average surface potential is about -4mV.
  • Each 1mg of PLGA nanovaccine is loaded with 90 ⁇ g of protein or peptide components.
  • Each 1mg of PLGA nanovaccine uses 0.048mg of adjuvant, of which CpG-ODN 1018 is 0.0056mg.
  • CpG-ODN M362 is 0.04mg
  • Poly(I:C) is 0.0024mg.
  • the average particle size of the control nanovaccine 1 is about 250nm, the average surface potential is about -5mV
  • each 1mg PLGA nanovaccine is loaded with 95 ⁇ g protein or peptide components
  • each 1mg PLGA nanovaccine uses 0.048mg adjuvant, of which CpG-ODN M362 is 0.047 mg
  • Poly(I:C) is 0.001mg (mass ratio is 47:1).
  • the average particle size of the control nanovaccine 2 is about 250nm, and the average surface potential is about -5mV.
  • Each 1 mg of PLGA nanovaccine is loaded with 90 ⁇ g of protein or peptide components and does not contain any adjuvants.
  • mice Female BALB/c mice aged 6-8 weeks were selected to prepare 4T1 tumor-bearing mice. On day 0, 4 ⁇ 10 5 4T1 cells were subcutaneously inoculated into the lower right side of the back of each mouse.
  • the vaccine treatment group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanovaccine on the 4th, 7th, 10th, 15th and 20th days.
  • the PBS blank control group was injected subcutaneously with 200 ⁇ L PBS on the 4th, 7th, 10th, 15th and 20th days respectively.
  • the blank nanoparticle + free lysate control group was subcutaneously injected with equal amounts of tumor tissue lysate and 2 mg PLGA blank nanoparticles on days 4, 7, 10, 15 and 20, respectively.
  • the mouse tumor volume monitoring and calculation methods were the same as above.
  • the tumor growth rate and mouse survival time in the vaccine treatment group were significantly different.
  • the tumors in mice in the vaccine group partially disappeared after vaccination.
  • the preventive effect of the nanovaccine with a total CpG-ODN and Poly(I:C) mass ratio of 19:1 as an adjuvant is better than that of a nanovaccine with a total CpG-ODN and Poly(I:C) mass ratio of 47:1 as an adjuvant. and vaccines without any adjuvants.
  • Example 12 Melanoma tumor tissue and whole cell components of lung cancer cells are loaded inside and on the surface of nano vaccines for the treatment of lung cancer
  • This example illustrates how to prepare a nanovaccine loaded with whole cell components of melanoma tumor tissue and lung cancer cancer cells, and use the vaccine to treat lung cancer.
  • B16F10 melanoma tumor tissue and LLC cancer cells were first lysed to prepare corresponding water-soluble components and non-water-soluble components dissolved in 8M urea aqueous solution (containing 500mM sodium chloride).
  • the water-soluble components from the tumor tissue and the water-soluble components from the cancer cells were mixed at a mass ratio of 1:1 to obtain the water-soluble components used in the experiment; the non-water-soluble components from the tumor tissue and the water-soluble components from the cancer cells were mixed The non-water-soluble components of cells are mixed at a mass ratio of 1:1 to be the non-water-soluble components used in the experiment; then, PLGA is used as the skeleton material, Poly(I:C), CpG-ODN 1018 (Class B) and CpG-ODN 1826 (category B) as immune adjuvants to prepare nano vaccines.
  • the lysis method of B16F10 melanoma tumor tissue and LLC lung cancer cells is the same as above. After the melanoma tumor tissue or LLC cancer cells are lysed, the lysate is centrifuged at 8000g for 5 minutes and the supernatant is taken to be soluble in pure water.
  • Water-soluble components Dissolve the precipitated part with 8M urea aqueous solution (containing 500mM sodium chloride) to convert the non-water-soluble components insoluble in pure water into soluble in 8M urea aqueous solution (containing 500mM sodium chloride). Water-soluble components and non-water-soluble components are mixed in a mass ratio of 1:1 respectively to obtain a water-soluble component mixture and a water-insoluble component mixture, which are the sources of raw materials for vaccine preparation.
  • the nanovaccine and blank nanoparticles are prepared using an appropriately improved double emulsion method.
  • the water-soluble component mixture is loaded inside the nanoparticles, but not the water-soluble component mixture is loaded on the surface of the nanovaccine.
  • the nanoparticle preparation material used is PLGA.
  • the molecular weight is 7KDa-17KDa
  • the immunoadjuvant used is Poly(I:C), CpG-ODN 1018 and CpG-ODN 1826 distributed inside and on the surface of the nanoparticles.
  • two modification methods, low-temperature siliconization technology and addition of charged substances, were used to increase the loading capacity of the antigen.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load water-soluble components and adjuvants inside the nanoparticles. After loading the antigen and adjuvants internally, 100mg nanoparticles are centrifuged at 10000g for 20 minutes, and then 7mL PBS is used. The nanoparticles were resuspended and mixed with 3 mL of PBS solution containing a mixture of water-insoluble components (60 mg/mL), followed by centrifugation at 10,000 g for 20 minutes, and then 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate) was used.
  • the non-water-soluble component of the immune adjuvant (1 mg/mL) (protein concentration 50 mg/mL) was reacted at room temperature for 10 minutes to obtain a siliconized and modified nanoparticle system loaded with lysate components both inside and outside.
  • the average particle size of the nanoparticles is about 350nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLGA nanoparticles is loaded with approximately 250 ⁇ g of protein or peptide components, and the immune adjuvant used per 1 mg of PLGA nanoparticles is 0.05 mg, of which Poly(I :C) is 0.01mg, CpG-ODN 1018 (Category B) is 0.03mg and CpG-ODN 1826 (Category B) is 0.01mg.
  • the preparation method of the control nanovaccine is the same as above.
  • the average particle size of the nanoparticles is about 350nm, and the surface potential of the nanoparticles is about -4mV.
  • Each 1 mg PLGA nanoparticle is loaded with approximately 250 ⁇ g of protein or peptide components.
  • the immune adjuvant used per 1 mg PLGA nanoparticle is: 0.05mg, of which Poly(I:C) is 0.01mg, CpG-ODN 1585 (Class A) is 0.03mg and CpG-ODN 2216 (Class A) is 0.01mg.
  • the particle size of the blank nanoparticles is about 320nm.
  • the blank nanoparticles contain equal amounts of Poly(I:C), CpG-ODN 1018 and CpG-ODN 1826, but do not contain lysate components.
  • Lung cancer tumor-bearing mice were prepared from female C57BL/6 mice aged 6 to 8 weeks. On day 0, each mouse was subcutaneously inoculated with 1 ⁇ 10 6 LLC lung cancer cells on the lower right side of the back.
  • the vaccine treatment group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanovaccine on the 4th, 7th, 10th, 15th and 20th days.
  • the PBS blank control group was injected subcutaneously with 200 ⁇ L PBS on the 4th, 7th, 10th, 15th and 20th days respectively.
  • the blank nanoparticle + free lysate control group was subcutaneously injected with equal amounts of tumor tissue lysate and 2 mg PLGA blank nanoparticles on days 4, 7, 10, 15 and 20, respectively.
  • the mouse tumor volume monitoring and calculation methods were the same as above.
  • Example 13 Colon cancer tumor tissue whole cell components loaded into nano vaccines for the treatment of colon cancer
  • This example illustrates how to prepare a nanovaccine loaded with whole cell components of colon cancer tumor tissue and use the vaccine to treat colon cancer.
  • MC38 colon cancer tumor tissue was first lysed to prepare corresponding water-soluble components and non-water-soluble components dissolved in 8M urea.
  • the water-soluble components and non-water-soluble components from tumor tissues are mixed at a mass ratio of 1:1 to form the lysate components used in the experiment; then, PLGA is used as the skeleton material, Poly(I:C), CpG -ODN 2395 (Category C) and CpG-ODN SL03 (Category C) are used as immune adjuvants, and NH 4 HCO 3 is used as a substance that increases lysosomal escape to prepare nano vaccines.
  • mice Female C57BL/6 mice aged 6 to 8 weeks were selected and inoculated with 2 ⁇ 10 6 MC38 colon cancer cells on the back. When the tumors grew to a volume of approximately 1000 mm3, the mice were sacrificed and the tumor tissues were removed. The tumor tissue was cut into pieces and then ground. An appropriate amount of pure water was added through a cell filter, treated with ultraviolet irradiation and heating at 45°C, and repeated freezing and thawing 5 times, accompanied by ultrasound to destroy the lysed tissue cells.
  • tissue cells After the tissue cells are lysed, centrifuge the lysate at 8000g for 5 minutes and take the supernatant, which is the water-soluble component that is soluble in pure water; add 8M urea to the resulting precipitate to dissolve the precipitate and remove the insoluble components.
  • the water-insoluble components of pure water are converted into soluble in 8M urea aqueous solution.
  • the nanovaccine and blank nanoparticles are prepared using an appropriately improved double emulsion method.
  • the water-soluble component mixture is loaded inside the nanoparticles, but not the water-soluble component mixture is loaded on the surface of the nanovaccine.
  • the nanoparticle preparation material used is PLGA.
  • the molecular weight is 7KDa-17KDa.
  • the immune adjuvant used is Poly(I:C), CpG-ODN 2395 and CpG-ODN SL03 distributed inside and on the surface of the nanoparticles, and NH 4 HCO 3 is wrapped inside the nanovaccine.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysate components and adjuvants inside the nanoparticles.
  • nanoparticles After loading the antigen and adjuvants inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes, and 10 mL containing 4 % trehalose in ultrapure water and freeze-dried for 48 hours before use. Before use, resuspend the particles in 9 mL of PBS and then add 1 mL of non-water-soluble components (protein concentration 50 mg/mL) containing immune adjuvant (1 mg/mL) and incubate at room temperature for 10 min before use.
  • protein concentration 50 mg/mL protein concentration 50 mg/mL
  • immune adjuvant 1 mg/mL
  • the average particle size of the nanoparticles is about 250nm, and the surface potential of the nanoparticles is about -4mV; each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or polypeptide components, and the immune adjuvant used per 1 mg of PLGA nanoparticles is 0.04 mg, of which Poly(I :C) is 0.01mg, CpG-ODN 2395 (Category C) is 0.02mg and CpG-ODN SL03 (Category C) is 0.01mg, loading NH 4 HCO 3 0.05mg.
  • the preparation method of the control nanovaccine is the same as above.
  • the average particle size of the nanoparticles is about 250nm, and the surface potential of the nanoparticles is about -4mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components.
  • the immune adjuvant used for each 1 mg of PLGA nanoparticles is: 0.04mg, of which Poly(I:C) is 0.01mg, CpG-ODN 1585 (Class A) is 0.02mg and CpG-ODN 2336 (Class A) is 0.01mg, loading NH 4 HCO 3 0.05mg.
  • the particle size of the blank nanoparticles is about 240nm.
  • the blank nanoparticles contain equal amounts of Poly(I:C), CpG-ODN 2395 (Category C), CpG-ODN SL03 (Category C) and NH 4 HCO 3 , but do not contain lysates. components.
  • Colon cancer tumor-bearing mice were prepared from 6-8 week old female C57BL/6 mice. Each mouse was subcutaneously inoculated with 1 ⁇ 10 6 MC38 colon cancer cells on the lower right side of the back on day 0.
  • the vaccine treatment group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanovaccine on the 4th, 7th, 10th, 15th and 20th days.
  • the PBS blank control group was injected subcutaneously with 200 ⁇ L PBS on the 4th, 7th, 10th, 15th and 20th days respectively.
  • the blank nanoparticle + free lysate control group was subcutaneously injected with equal amounts of tumor tissue lysate and 2 mg PLGA blank nanoparticles on days 4, 7, 10, 15 and 20, respectively.
  • the mouse tumor volume monitoring and calculation methods were the same as above.
  • Example 14 Water-soluble cell components loaded inside and on the surface of microparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a micron vaccine loaded only with the water-soluble part of melanoma and lung cancer cell components, and use the vaccine to prevent melanoma.
  • B16F10 melanoma and LLC lung cancer cells were first lysed to prepare water-soluble components and non-water-soluble components. Then, use PLGA (24KDa-38KDa) as the micron particle skeleton material, poly(I:C) and two CpG-ODN as immune adjuvants to prepare micron vaccines loaded with water-soluble components of whole cells, and use the micron Vaccines prevent melanoma.
  • B16F10 cells or LLC cells Collect a certain amount of B16F10 cells or LLC cells, remove the culture medium and freeze them at -20°C. Add a certain amount of ultrapure water and process the cells by heating and ultraviolet irradiation. Then freeze and thaw repeatedly for more than 3 times, accompanied by ultrasonic damage to lyse the cells. . After the cells are lysed, centrifuge the lysate at 8000g for 5 minutes and take the supernatant, which is the water-soluble component of B16F10 melanoma or LLC lung cancer cells that is soluble in pure water. The water-soluble components derived from the two cancer cell lysates obtained above are mixed at a mass ratio of 1:1 to form the antigen source for preparing micron vaccines.
  • the double emulsion method in the solvent evaporation method was used to prepare micron vaccines and blank micron particles as controls.
  • the micron particle preparation material used was PLGA, and the immune adjuvants used were poly(I:C), CpG- ODN 2006 (Category B) and CpG-ODN2216 (Category A), the mass ratio of the three is 4:0.5:0.5.
  • the antigen component is contained inside the micron vaccine and adsorbed on the surface of the micron particles, while the adjuvant is only contained inside the micron vaccine.
  • the preparation method is as described above.
  • the particle size of the micron vaccine obtained after loading cell components and immune adjuvants on the surface of the micron particles is 2.10 ⁇ m, and the surface potential is about -5mV.
  • Each 1 mg of PLGA micron particles is loaded with 150 ⁇ g of protein or peptide components, and 0.05 mg of adjuvant is used, where Poly(I:C)0.04mg, CpG-ODN 2006 0.005mg, CpG-ODN 2216 0.005mg.
  • the control micron vaccine 1 has a particle size of 2.10 ⁇ m and a surface potential of -5mV.
  • Each 1 mg PLGA micron particle is loaded with 150 ⁇ g of protein or peptide components.
  • Poly(I:C) and CpG-ODN2006 are used as adjuvants, in which poly(I:C )0.04mg, CpG-ODN 2006 0.01mg.
  • Control micron vaccine 2 has a particle size of 2.10 ⁇ m and a surface potential of -5mV. Each 1mg PLGA micron particle is loaded with 150 ⁇ g protein or peptide components.
  • Poly(I:C), CpG-ODN 2006 and CpG-ODN 2216 are used as adjuvants. Among them poly(IC)0.025mg, CpG-ODN 2006 0.0125mg, CpG-ODN 2216 0.0125mg.
  • Melanoma tumor-bearing mice were prepared from 6-8 week old female C57BL/6 mice.
  • the plan for the micron vaccine group is as follows: 200 ⁇ L of 2 mg PLGA micron vaccine was subcutaneously injected on the 35th, 28th, 21st, and 14th days before melanoma inoculation; on the 0th day, each mouse was subcutaneously inoculated on the lower right side of the back. 1.5 ⁇ 10 5 B16F10 cells.
  • the plan for the PBS blank control group is as follows: subcutaneously inject 200 ⁇ L PBS on the 35th, 28th, 21st, and 14th days before inoculation of melanoma; on the 0th day, subcutaneously inoculate 1.5 ⁇ 10 5 B16F10 cells.
  • the tumor volume growth rate of mice in the micron vaccine administration group was significantly slower and the survival period of mice was significantly prolonged.
  • the Micron vaccine using poly(I:C) and two CpG-ODN as mixed adjuvants was better than the Micron vaccine using only Poly(I:C) and one CpG-ODN as mixed adjuvants; using total CpG-
  • Example 15 Adjuvants and melanoma cancer cells are loaded inside nanoparticles for the treatment of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nanovaccine co-loaded with an immune adjuvant and whole cell components of melanoma cancer cells, and use the vaccine to treat melanoma.
  • B16F10 mouse melanoma cells were used as the cancer model.
  • B16F10 melanoma cancer cells were first lysed and water-soluble and water-insoluble fractions were prepared. Then, the organic polymer material PLGA was used as the nanoparticle skeleton material, poly(I:C), CpG-ODN 1018, CpG-ODN 2336 and arginine were used as immune adjuvants to prepare water-soluble cancer cells loaded with the solvent evaporation method.
  • Nanovaccines with water-soluble and non-water-soluble components The nanovaccine is then used to treat melanoma.
  • the cultured B16F10 cancer cell line was removed from the culture medium, centrifuged at 400g for 5 minutes, resuspended in PBS and washed twice, then resuspended the cancer cells in ultrapure water, and repeatedly frozen and thawed five times. Ultrasound was used to assist during the freezing and thawing process.
  • the cancer cells were lysed, and then centrifuged at 10,000 g for 5 minutes, and the supernatant was collected as the water-soluble fraction. Dissolve the precipitated part with 8M urea, which is the dissolved original non-water-soluble component. Mixing the water-soluble components from B16F10 cancer cells and the original non-water-soluble components dissolved in 8M urea at a mass ratio of 1:1 is the source of raw materials for vaccine preparation.
  • the nano vaccine is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nano particle preparation material PLGA used is 7KDa-17 KDa.
  • the adjuvant and antigen components are contained in the nano vaccine.
  • the immune adjuvant used It is poly(I:C), CpG-ODN 1018, CpG-ODN 2336 and arginine.
  • the preparation method is as described above.
  • the average particle size of nanovaccines loaded with whole cell components is about 280nm; the surface potential of nanovaccines is about -5mV; each 1mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components, and the adjuvant used per 1mg of PLGA nanoparticles is 0.05mg.
  • poly(I:C) is 0.02mg
  • CpG-ODN 1018 is 0.01mg
  • CpG-ODN 2336 is 0.02mg
  • arginine is 0.5mg.
  • the preparation method of the control nanovaccine 1 is the same, with a particle size of 280nm and a vaccine surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components; the adjuvant used for each 1 mg of PLGA nanoparticles is 0.05 mg, poly(I :C) is 0.02mg, CpG-ODN 1018 is 0.015mg, and CpG-ODN 2336 is 0.015mg.
  • control nanovaccine 2 The preparation method of control nanovaccine 2 is the same, with a particle size of 280nm and a vaccine surface potential of about -5mV; each 1 mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein or peptide components; each 1 mg PLGA nanoparticle uses 0.5 mg arginine as an immune adjuvant. .
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice.
  • the dosing schedule of the nanovaccine group is as follows: 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of each mouse’s back on day 0; on days 4, 7, 10, and 15 after inoculation with melanoma and on the 20th day, 100 ⁇ L of 2 mg PLGA nanovaccine loaded with water-soluble components was injected subcutaneously.
  • the plan for the PBS control group is as follows: 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse on day 0; on the 4th, 7th, 10th, 15th, and On the 20th day, 200 ⁇ L PBS was injected subcutaneously.
  • the mouse tumor monitoring protocol is the same as above.
  • the tumors of mice in the PBS control group all grew larger and grew rapidly.
  • the efficacy of the nanovaccine group was better than that of the PBS control group, and nanovaccines using two or more CpG+Poly(I:C)+arginine as mixed adjuvants were better than those using two or more CpG+Poly(I:C) Nanovaccines that are mixed adjuvants are also better than nanovaccines that only use arginine as an adjuvant.
  • the nano-vaccine loaded with specific proportions of multiple CpGs, Poly(I:C) and antigen components has good therapeutic effect on melanoma.
  • Example 16 Adjuvants and breast cancer cancer cells are loaded inside nanoparticles for the treatment of breast cancer
  • This example uses mouse triple-negative breast cancer as a cancer model to illustrate how to prepare a nanovaccine that co-loads immune adjuvants, substances that increase lysosomal escape, and whole-cell components of triple-negative breast cancer cells, and uses this vaccine to treat Triple negative breast cancer.
  • 4T1 breast cancer cancer cells are first lysed and water-soluble components and water-insoluble components are prepared. Then, PLGA was used as the nanoparticle skeleton material, Poly(I:C), CpG-ODN 1018, and CpG-ODN 2336 were used as immune adjuvants, and lysine was used as a substance to increase lysosomal escape, and the solvent evaporation method was used to prepare the load. Nano-vaccines with water-soluble components and non-water-soluble components of cancer cells. The nanovaccine is then used to treat breast cancer.
  • the nano vaccine is prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nano particle preparation material PLGA used is 7KDa-17 KDa.
  • the adjuvant and antigen components are contained in the nano vaccine.
  • the immune adjuvant used It is poly(I:C), CpG-ODN 1018, CpG-ODN 2336 and lysine.
  • the preparation method is as described above.
  • the average particle size of the nanovaccine loaded with whole cell components is about 280nm; the surface potential of the nanovaccine is about -5mV; each 1mg PLGA nanoparticle is loaded with approximately 90 ⁇ g of protein or peptide components, and the adjuvant used per 1mg PLGA nanoparticle is 0.05mg , where poly(I:C) is 0.02mg, CpG-ODN 1018 is 0.015mg, CpG-ODN 2216 is 0.015mg, and lysine is used 0.005mg.
  • the preparation method of the control nanovaccine 1 is the same, with a particle size of 280nm and a vaccine surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components; the adjuvant used per 1 mg of PLGA nanoparticles is 0.05 mg, of which poly (I:C) is 0.02mg, CpG-ODN 1018 is 0.015mg, CpG-ODN 2216 is 0.015mg, and 0.005mg of glycine is used.
  • the preparation method of the control nanovaccine 2 is the same, with a particle size of 280nm and a vaccine surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components; the adjuvant used for each 1 mg of PLGA nanoparticles is 0.05 mg, in which poly( I:C) is 0.02mg, CpG-ODN 1585 is 0.015mg, CpG-ODN 2216 is 0.015mg, and lysine 0.005mg is used.
  • mice Female BALB/c mice aged 6-8 weeks were selected as model mice to prepare breast cancer tumor-bearing mice.
  • the dosing schedule of the nanovaccine group is as follows: 4 ⁇ 10 5 4T1 cells were subcutaneously inoculated into the lower right corner of each mouse's back on day 0; on days 4, 7, 10, and 15 after breast cancer inoculation and on the 20th day, 100 ⁇ L of 2 mg PLGA nanovaccine loaded with water-soluble components was injected subcutaneously.
  • the plan for the PBS control group is as follows: on day 0, 4 ⁇ 10 5 4T1 cells were subcutaneously inoculated into the lower right corner of the back of each mouse; on the 4th, 7th, 10th, 15th, and On the 20th day, 200 ⁇ L PBS was injected subcutaneously.
  • the mouse tumor monitoring protocol is the same as above.
  • the tumors of mice in the PBS control group all grew larger and produced rapidly.
  • the efficacy of the nanovaccine group was better than that of the PBS control group, and the nanovaccine using two or more CpG-ODN (type A + type B) + Poly (I:C) + lysine as a mixed adjuvant was better than using two kinds of nanovaccines.
  • the above CpG-ODN (Type A + Type B) + Poly (I:C) nanovaccine as a mixed adjuvant is also better than using two or more CpG-ODN (Type A + Type A) + Poly (I:C) + Lysine as an adjuvant for nanovaccines.
  • the nano-vaccine loaded with specific proportions of multiple CpGs, Poly(I:C) and antigen components has a good therapeutic effect on breast cancer.
  • Example 17 Whole cell components loaded inside mannan-modified micron particles for breast cancer prevention
  • This example uses mouse breast cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with whole cell components of breast cancer tumor tissue and cancer cells, and apply the vaccine to prevent breast cancer.
  • particle size, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • tumor tissue and cancer cells of mouse breast cancer were first obtained and lysed to prepare water-soluble components and original water-insoluble components dissolved in 8M urea.
  • micron vaccines are prepared using the solvent evaporation method. TAT polypeptide contains positively charged lysine and arginine. The micron vaccine has the ability to target dendritic cells.
  • the mice are sacrificed and the tumor tissue is removed.
  • the methods for lysing 4T1 cancer cells and cancer tumor tissues and collecting each component are the same as above.
  • the micron vaccine and the empty micron particles used as controls were prepared by the double emulsion method.
  • the molecular weight of the microparticle preparation material PLGA is 24KDa-38KDa, and the molecular weight of the mannan-modified PLGA used is 24KDa-38KDa; the mass ratio of unmodified PLGA and mannan-modified PLGA is 8:2.
  • the preparation method is as described above, and the lysate components and adjuvants are co-loaded into the micron vaccine.
  • the average particle size of the micron vaccine is about 1.50 ⁇ m, and the average surface potential is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA micron vaccine uses 0.05 mg of adjuvant, of which Poly ICLC 0.01 mg, CpG- ODN 1018 (Category B) 0.02mg, CpG-ODN 2395 (Category C) 0.02mg, TAT peptide 0.1mg.
  • the polymer materials and preparation methods used in the preparation of blank microparticles are the same as above.
  • the particle size is about 1.45 ⁇ m.
  • the average particle size is about 1.50 ⁇ m, the average surface potential is about -7mV, each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components, and each 1 mg of PLGA micron vaccine uses an adjuvant.
  • the tumor growth rate and mouse survival time in the vaccine prevention group were significantly different.
  • the micron vaccine prepared in this example is better than the control micron vaccine group, which shows that using a mixture of Class C CpG-ODN and Class B CpG-ODN as an adjuvant is more effective than mixing two Class A CpG-ODN.
  • Example 18 Liver cancer tumor tissue lysis components loaded on micron particles for preventing liver cancer
  • a micron vaccine loaded with liver cancer and lung cancer tumor tissue lysate components is prepared, and the vaccine is used to prevent liver cancer.
  • the mouse liver cancer tumor tissue lysate components were loaded into the micron vaccine.
  • mouse liver cancer and lung cancer tumor tissues were obtained, and 8M urea was used to lyse the tumor tissues and dissolve the tumor tissue lysate components.
  • PLA (30KDa) was used as the micron particle framework material
  • Poly ICLC, CpG-ODN 2007 (Class B) and CpG-ODN2216 (Class A) were used as immune adjuvants
  • cR8 (cyclo (CRRRRRRRRC)) polypeptide was used to increase immunity. escape substances, prepare micron vaccines, and apply the micron vaccines to prevent liver cancer.
  • cR8 is a cyclic polypeptide containing arginine.
  • mice Each C57BL/6 mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 cells or 2 ⁇ 10 6 LLC lung cancer cells under the armpit, and each mouse was sacrificed when the inoculated tumor grew to a volume of approximately 1000 mm 3 . mice and remove tumor tissue. The tumor tissue was minced and passed through a cell sieve, and then 8M urea was used to lyse the tumor tissue and then dissolve; the liver cancer tumor tissue lysate and the lung cancer tumor tissue lysate were mixed at a mass ratio of 1:1 to form the antigen component used for vaccine preparation.
  • the micron vaccine is prepared by a solvent evaporation method.
  • the preparation material used is PLA.
  • the immune adjuvants used are Poly ICLC, CpG-ODN 2007, and CpG-ODN 2216. The mass ratio of the four is 1:1:1;
  • the substance used to increase lysosome escape is cR8 polypeptide.
  • One-fifth of the cR8 polypeptide is modified with PLA through chemical bonds and is located on the surface of the particle, while four-fifths of the cR8 polypeptide is contained inside the particle.
  • the antigen components are contained inside the micron vaccine and adsorbed on the surface of the micron particles.
  • the adjuvants and substances that increase lysosome escape are only contained inside the micron vaccine.
  • the antigen components and adjuvants are loaded inside the microparticles using the dissolution and evaporation method, then centrifuged at 8000g for 15 minutes, the precipitate is collected and 100mg of PLA microparticles are resuspended in 4% trehalose solution, then freeze-dried for 48 hours and refrigerated. spare.
  • 10 mg PLA micron particles are resuspended in 0.9 mL PBS or normal saline, and then mixed with 0.1 mL of cell lysate (60 mg/mL) containing 8 M urea and immune adjuvant (3 mg/mL, the mass ratio of the four 1:1:1) sample is mixed at room temperature for 10 minutes and is ready for injection.
  • the particle size of the obtained micron vaccine is 3.10 ⁇ m, and each 1 mg PLA micron particle is loaded with 150 ⁇ g of protein or peptide components.
  • An adjuvant of 0.045 mg is used, including Poly ICLC 0.015 mg, CpG-ODN2007 0.015 mg, and CpG-ODN 2216 0.015 mg; cR8 polypeptide is used 0.02mg, of which 0.004mg is connected to PLA through chemical modification and is located on the surface of the particles, and 0.016mg is contained inside the particles.
  • the particle size of the control micron vaccine is 3.10 ⁇ m, and each 1 mg PLA micron particle is loaded with 150 ⁇ g of protein or peptide components.
  • Poly ICLC, CpG-ODN 1585 and CpG-ODN 2216 are used at 0.015 mg each; cR8 peptide is used at 0.02 mg, of which 0.004 mg passes
  • the chemical modification is connected to the PLA on the surface of the particles, and 0.016mg is contained inside the particles.
  • the particle size of 2 blank micron particles loaded with immune adjuvants for the control micron vaccine is 3.00 ⁇ m.
  • Each 1 mg PLA micron particle is loaded with 150 ⁇ g of protein or peptide components.
  • Poly ICLC 0.015 mg, CpG-ODN 2007 0.03 mg, and cR8 peptide 0.02 mg are used. , of which 0.004mg is connected to PLA through chemical modification and is located on the surface of the particles, and 0.016mg is contained inside the particles.
  • Hepa 1-6 liver cancer tumor-bearing mice were prepared from 6-8 week old female C57BL/6 mice. 200 ⁇ L of 2 mg PLA micron vaccine was injected subcutaneously on days 49, 42, 35, 28, and 14 before inoculation of liver cancer cells. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit.
  • the protocol for the PBS blank control group is as follows: 200 ⁇ L PBS was injected subcutaneously on the 49th, 42nd, 35th, 28th and 14th days before inoculation of liver cancer cells. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit. In the experiment, the mouse tumor growth monitoring method was the same as above.
  • the liver cancer tumors of mice in the PBS control group all grew faster, and the micron vaccine administration group could significantly inhibit mouse tumor growth and prolong the survival of mice.
  • the vaccine used in this example is more effective than Control Vaccine 1 and Control Vaccine 2. It can be seen that the simultaneous use of Class B CpG-ODN and Class A CpG-ODN and Poly ICLC as mixed adjuvants is better than the use of only Class B CpG-ODN and Poly ICLC as mixed adjuvants and the simultaneous use of two Class A CpG-ODNs. With Poly ICLC as mixed adjuvant.
  • Example 19 Tumor tissue lysis components loaded inside nanoparticles for the treatment of pancreatic cancer
  • mouse pancreatic cancer as a cancer model to illustrate how to prepare a nanovaccine loaded with pancreatic cancer tumor tissue lysate components and use the vaccine to treat pancreatic cancer.
  • mouse pancreatic cancer tumor tissue was obtained and lysed to prepare water-soluble fractions and original non-water-soluble fractions dissolved in 6M guanidine hydrochloride.
  • PLGA molecular weight 7KDa-17KDa
  • Poly(I:C) Poly(I:C)
  • CpG-ODN 2336 immune adjuvants
  • HER2 polypeptide YDLKEPEH
  • HER2 polypeptide contains histidine and arginine.
  • Pan02 pancreatic cancer cells were subcutaneously inoculated into the armpit of each C57BL/6 mouse. When the inoculated tumors in each mouse grew to a volume of approximately 1000 mm 3 , the mice were sacrificed and the tumor tissues were harvested. The tumor tissue lysis method is the same as in Example 18. After collecting the water-soluble components, 6M guanidine hydrochloride is used to dissolve the non-water-soluble components.
  • the solvent evaporation method is used to prepare the nano-vaccine.
  • the molecular weight of PLGA is 24KDa-38KDa.
  • Poly(I:C), CpG-ODN M362, and CpG-ODN 2336 are used as mixed adjuvants in a mass ratio of 1:0.8:0.8.
  • HER2 polypeptide is used as a substance to increase lysosomal escape.
  • the particle size of the nanovaccine is about 270nm, and the surface potential is about -7mV.
  • Each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components.
  • a total of 0.026 mg of adjuvant is used for each 1 mg of PLGA nanoparticles, of which Poly(I:C) is 0.01. mg, CpG-ODN M362 is 0.008mg: CpG-ODN 2336 is 0.008mg; use HER2 peptide 0.026mg.
  • the control nanovaccine has a particle size of about 280nm and a surface potential of about -8mV.
  • Each 1mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein or peptide components.
  • Each 1mg of PLGA nanoparticles uses a total of 0.026mg of adjuvants, of which Poly(I:C) is 0.01mg, CpG-ODN M362 is 0.08mg: CpG-ODN 2336 is 0.08mg.
  • the particle size of the control nanovaccine 2 is about 280nm, and the surface potential is about -8mV.
  • Each 1mg PLGA nanoparticle is loaded with approximately 80 ⁇ g of protein or peptide components.
  • Each 1mg PLGA nanoparticle uses a total of 0.026mg of adjuvant.
  • Poly(I:C) is 0.01mg, of which CpG1585 is 0.08mg, CpG-ODN 2216 is 0.08mg, and 0.026mg of HER2 peptide is used.
  • mice Female C57BL/6 mice aged 6-8 weeks were selected to prepare pancreatic cancer mice. On day 0, 1 ⁇ 10 6 Pan02 cells were subcutaneously inoculated into the lower right corner of the back of each mouse.
  • the vaccine group was subcutaneously injected with 100 ⁇ L of 1 mg PLGA nanovaccine loaded with water-soluble components and 100 ⁇ L of 1 mg PLGA nanovaccine loaded with original non-water-soluble components on the 4th, 7th, 10th, 15th and 20th days.
  • the PBS blank control group was injected subcutaneously with 200 ⁇ L PBS on the 4th, 7th, 10th, 15th and 20th days respectively.
  • the mouse tumor monitoring and volume calculation methods were the same as above.
  • the nanovaccine according to the present invention loaded with a specific proportion of a variety of CpG and Poly(I:C) mixed adjuvants and antigen components has a good therapeutic effect on cancer, and it also contains positively charged amino acids.
  • Peptides can improve vaccine efficacy, and mixing one type C CpG-ODN and one type A CpG-ODN is more effective than using two type A CpG-ODNs.
  • Example 20 Nanovaccine exerts cancer therapeutic effect through T cell immunity in cellular immunity
  • This example uses mouse melanoma as a cancer model to illustrate that the nanovaccine mainly exerts its effect through T cell immunity in cellular immunity.
  • B16F10 melanoma cancer cells were first lysed and water-soluble components and water-insoluble components were prepared. Then, PLGA was used as the nanoparticle skeleton material, poly(I:C), CpG-ODN 1018 (B type), CpG-ODN 2336 (A type) as immune adjuvants, and arginine and polyhistidine as the immune adjuvants.
  • a nanovaccine loaded with water-soluble components and non-water-soluble components of cancer cells is prepared using a solvent evaporation method. The nanovaccine is then used to treat melanoma.
  • the cultured B16F10 cancer cell line was removed from the culture medium, centrifuged at 400g for 5 minutes, resuspended in PBS and washed twice, then resuspended the cancer cells in ultrapure water, and repeatedly frozen and thawed five times. Ultrasound was used to assist during the freezing and thawing process.
  • the cancer cells were lysed, and then centrifuged at 10,000 g for 5 minutes, and the supernatant was collected as the water-soluble fraction. Dissolve the precipitated part with 8M urea, which is the dissolved original non-water-soluble component. Mixing the water-soluble components from B16F10 cancer cells and the original non-water-soluble components dissolved in 8M urea at a mass ratio of 1:1 is the source of raw materials for vaccine preparation.
  • the nano vaccine is prepared by solvent evaporation method.
  • the molecular weight of PLGA used is 7KDa-17KDa.
  • the adjuvant, cleavage component, arginine and polyhistidine are contained in the nano vaccine.
  • the immune adjuvant used is poly(I:C), CpG-ODN 1018, CpG-ODN 2336, the substances that increase immune evasion are arginine and polyhistidine.
  • the preparation method is as described above.
  • the average particle size of nanovaccines loaded with whole cell components is about 280nm; the surface potential of nanovaccines is about -8mV; each 1mg of PLGA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components, and the adjuvant used per 1mg of PLGA nanoparticles is 0.05mg.
  • poly(I:C) is 0.02mg
  • CpG-ODN 1018 is 0.015mg
  • CpG-ODN 2336 is 0.015mg
  • loaded arginine is 0.005mg
  • loaded polyhistidine is 0.005mg.
  • Melanoma tumor-bearing mice were prepared by selecting 6-8 week old female C57BL/6 as model mice.
  • the dosing schedule of the nanovaccine group is as follows: 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of each mouse’s back on day 0; on days 4, 7, 10, and 15 after inoculation with melanoma and 100 ⁇ L of 2 mg PLGA nanovaccine were subcutaneously injected on day 20; the CD4 + T cell antagonist group, in addition to the vaccine, was intraperitoneally injected with 10 mg/kg ⁇ CD4 antibody every day starting from the two days before day 0 of inoculation of cancer cells to eliminate mice.
  • CD4 + T cells in the body were continuously given antibodies until the 50th day; in addition to the vaccine, the CD8 + T cell antagonist group was intraperitoneally injected with 10 mg/kg ⁇ CD8 antibody every day starting two days before the 0th day of inoculation of cancer cells to eliminate them.
  • CD8 + T cells in mice were continuously given antibodies until day 50.
  • the plan for the PBS control group is as follows: 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated into the lower right corner of the back of each mouse on day 0; on the 4th, 7th, 10th, 15th, and On the 20th day, 200 ⁇ L PBS was injected subcutaneously.
  • the mouse tumor monitoring protocol is the same as above.
  • the tumors of the mice in the PBS control group grew up and were produced very quickly.
  • the tumor growth rate and survival period of the mice in the vaccine group were significantly better than those in the PBS control group.
  • the cancer vaccine After using ⁇ CD4 antibodies to deplete CD4 + T cells in mice, the cancer vaccine still has a strong therapeutic effect on cancer; after using ⁇ CD8 antibodies to deplete CD8 + T cells in mice, the cancer vaccine loses most of its efficacy and is only effective against cancer. Has a weak therapeutic effect. This shows that the vaccine of the present invention mainly exerts its effect through T cell immunity in cellular immunity.
  • mouse breast cancer as a cancer model to illustrate that the micron vaccine mainly works through T cells.
  • mouse breast cancer tumor tissue was first obtained, lysed with 8M urea and then dissolved in 8M urea. Then, PLGA and mannose-modified PLGA were used as micron particle framework materials, and Poly(I:C), CpG-ODN 1018 (B type) and CpG-ODN 2216 (A type) were used as immune adjuvants to prepare by solvent evaporation method.
  • Micron vaccine used as a cancer model to illustrate that the micron vaccine mainly works through T cells.
  • mouse breast cancer tumor tissue was first obtained, lysed with 8M urea and then dissolved in 8M urea. Then, PLGA and mannose-modified PLGA were used as micron particle framework materials, and Poly(I:C), CpG-ODN 1018 (B type) and CpG-ODN 2216 (A type) were used as immune adjuvants to prepare by solvent evapor
  • mice Inoculate 4 ⁇ 10 5 4T1 breast cancer cells into the armpit of each BALB/c mouse.
  • the mice are sacrificed and the tumor tissue is removed. Cut the tumor tissue into small pieces and filter it through a cell mesh to prepare a single-cell suspension.
  • 8M urea to lyse the cells and dissolve the lysed components, which are the antigen components for preparing micron vaccines.
  • the micron vaccine and the empty micron particles used as controls were prepared by the double emulsion method.
  • the molecular weight of PLGA, the material for preparing micron particles, is 38KDa-54KDa, and the molecular weight of the mannose-modified PLGA used is also 38KDa-54KDa.
  • the mass ratio of unmodified PLGA and mannose-modified PLGA was 8:2.
  • the preparation method is as described above, and the lysate components and adjuvant are co-loaded into the micron vaccine.
  • the preparation method is as described above.
  • the average particle size of the micron vaccine is about 2.50 ⁇ m, and the average surface potential is about -9mV.
  • Each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA micron vaccine uses 0.05 mg of adjuvant, of which CpG-ODN 1018 is 0.02 mg. , CpG-ODN 2216 is 0.01mg, Poly(I:C) is 0.002mg.
  • mice Begin to intraperitoneally inject 10 mg/kg of ⁇ CD8 antibody every day to eliminate CD8 + T cells in the mice, and continue to administer the antibody until the 50th day.
  • the PBS blank control group was subcutaneously injected with 200 ⁇ L PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation; on the 0th day, 4 ⁇ 10 mice were subcutaneously inoculated on the lower right side of the back of each mouse. 5 4T1 breast cancer cells.
  • the mouse tumor growth monitoring method was the same as above.
  • the tumor growth rate and mouse survival time in the vaccine prevention group were significantly different.
  • the cancer vaccine still has a strong preventive effect on cancer;
  • ⁇ CD8 antibodies to deplete CD8 + T cells in mice the cancer vaccine loses most of its efficacy and is only effective against cancer. Has a weak preventive effect. This shows that the vaccine of the present invention mainly exerts its effect through T cell immunity in cellular immunity.
  • Example 22 Whole cell components loaded on mannose-modified microparticles for breast cancer treatment
  • This example uses mouse breast cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with whole cell components of breast cancer tumor tissue and use the vaccine to treat breast cancer.
  • particle size, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • PLGA and mannose-modified PLGA are used as micron particle framework materials
  • Poly ICLC, CpG-ODN 1018 (B type) and CpG-ODN 2395 (C type) are used as immune adjuvants
  • arginine and Lysine mixture is used to increase immune evasion substances and prepare micron vaccines.
  • the micron vaccine has the ability to target dendritic cells.
  • the mice are sacrificed and the tumor tissue is removed.
  • the methods for lysing 4T1 cancer tumor tissue and collecting each component are the same as above.
  • the micron vaccine and the empty micron particles used as controls were prepared by the double emulsion method.
  • the molecular weight of the microparticle preparation material PLGA is 24KDa-38KDa, and the molecular weight of the mannose-modified PLGA used is 24KDa-38KDa; the mass ratio of unmodified PLGA and mannose-modified PLGA is 7:3.
  • the preparation method is as described above, and the lysate components and adjuvants are co-loaded into the micron vaccine.
  • the average particle size of the micron vaccine is about 1.50 ⁇ m, and the average surface potential is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA micron vaccine uses 0.05 mg of adjuvant, of which Poly ICLC 0.01 mg, CpG- ODN 1018 (Category B) 0.02mg, CpG-ODN 2395 (Category C) 0.02mg, use arginine 0.05mg, lysine 0.05mg.
  • the control micron vaccine 1 has a particle size of about 1.50 ⁇ m, an average surface potential of about -7mV, is loaded with equal amounts of adjuvants and cell lysate components, and is loaded with 0.1 mg arginine.
  • the average particle size of the control micron vaccine 2 is about 1.50 ⁇ m, and the average surface potential is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with 85 ⁇ g of protein or peptide components.
  • Each 1 mg of PLGA micron vaccine uses 0.05 mg of adjuvant, of which Poly ICLC is 0.01 mg. , CpG-ODN 1018 (Category B) 0.02mg, CpG-ODN 2395 (Category C) 0.02mg, loaded lysine 0.1mg.
  • the tumor growth rate and mouse survival time in the vaccine treatment group were significantly different.
  • the micron vaccine prepared in this example is better than the control micron vaccine group, which shows that the effect of using mixed amino acids is better than using single amino acids.
  • Example 23 Liver cancer tumor tissue lysis components loaded on micron particles for the treatment of liver cancer
  • a micron vaccine loaded with liver cancer tumor tissue lysate components is prepared, and the vaccine is used to prevent liver cancer.
  • PLGA 38KDa-54KDa
  • Poly ICLC Poly ICLC
  • CpG-ODN 2007 (Class B) and CpG-ODN2216 (Class A) are used as immune adjuvants
  • histidine is used as the dissolution-promoting agent.
  • Substances escaping from enzyme bodies are used to prepare micron vaccines, and the micron vaccines are used to treat liver cancer.
  • Hepa 1-6 liver cancer cells were subcutaneously inoculated into the armpit of each C57BL/6 mouse.
  • the mice were sacrificed and the tumor tissues were removed.
  • the tumor tissue was minced and passed through a cell sieve, and then 6M guanidine hydrochloride was used to lyse the tumor tissue.
  • the lysed component was then used to prepare the antigen component for the vaccine.
  • the micron vaccine is prepared by a solvent evaporation method.
  • the preparation material used is PLGA.
  • the immune adjuvants used are Poly ICLC, CpG-ODN 2007, and CpG-ODN 2216.
  • the mass ratio of the four is 1:1:1;
  • the substance used to increase lysosomal escape is histidine. Antigen components, adjuvants and substances that increase lysosomal escape are contained within the micron vaccine.
  • the antigen components and adjuvants are loaded inside the microparticles using the dissolution and evaporation method, then centrifuged at 8000g for 15 minutes, the precipitate is collected and 100mg of PLA microparticles are resuspended in 4% trehalose solution, then freeze-dried for 48 hours and refrigerated. spare.
  • the particle size of the obtained micron vaccine is 2.60 ⁇ m, and each 1 mg PLGA micron particle is loaded with 100 ⁇ g of protein or peptide components.
  • An adjuvant of 0.045 mg is used, including Poly ICLC 0.015 mg, CpG-ODN 2007 0.015 mg, and CpG-ODN 2216 0.015 mg; the use group Acid 0.02mg.
  • the particle size of the control micron vaccine is 2.60 ⁇ m, and each 1 mg PLGA micron particle is loaded with 100 ⁇ g of protein or peptide components.
  • Poly ICLC, CpG-ODN 2007, and CpG-ODN 2216 are used at 0.015 mg each; glutamic acid is used at 0.02 mg.
  • the particle size of the control micron vaccine 2 is 2.60 ⁇ m, and each 1 mg PLGA micron particle is loaded with 100 ⁇ g protein or peptide components.
  • Poly ICLC 0.015 mg, CpG-ODN 2007 0.015 mg, CpG-ODN 2216 0.015 mg are used; 8 histidine-containing Peptide (LHQAVVPGL) 0.02 mg.
  • Hepa 1-6 liver cancer tumor-bearing mice were prepared from 6-8 week old female C57BL/6 mice. On day 0, each mouse was subcutaneously inoculated with 2 ⁇ 10 6 Hepa 1-6 liver cancer cells in the right armpit. On days 4, 7, 10, 15, 20 and 25 after inoculation of liver cancer cells, 200 ⁇ L of 2 mg PLA micron vaccine was injected subcutaneously. In the experiment, the mouse tumor growth monitoring method was the same as above.
  • the liver cancer tumors of mice in the PBS control group all grew faster, and the micron vaccine administration group could significantly inhibit mouse tumor growth and prolong the survival of mice.
  • the vaccine used in this example is more effective than Control Vaccine 1 and Control Vaccine 2. It can be seen that the effect of using positively charged amino acids is better than that of negatively charged amino acids, and the effect of pure histidine is better than that of peptides containing part of histidine acid.

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Abstract

涉及一种免疫佐剂组合物和基于该组合物的癌症疫苗及其应用，免疫佐剂组合物至少包括以下组分中的(1)和(2)的组合，(1)Poly(I:C)或Poly(ICLC)，(2)CpG-ODN，其中，CpG-ODN为A类CpG-ODN、B类CpG-ODN和C类CpG-ODN中的至少两种，且至少其中一种为B类CpG-ODN或C类CpG-ODN，(3)氨基酸、多肽、脂类、糖类、蛋白质或无机盐。基于该免疫佐剂组合物提供了一种癌症疫苗，包括纳米粒子或微米粒子，以及负载于其上的抗原组分和免疫佐剂组合物。提供的免疫佐剂组合物可以充分发挥增强佐剂激活癌症特异性T细胞反应时的功效，更好地辅助疫苗发挥功能。

Description

一种免疫佐剂组合物和基于该组合物的癌症疫苗及其应用 技术领域
本发明涉及免疫治疗领域，尤其涉及一种免疫佐剂组合物和基于该组合物的癌症疫苗及其应用。
背景技术
癌症疫苗是癌症免疫治疗和预防的重要方法之一。癌症疫苗最重要的两部分是抗原和免疫佐剂，抗原可以提供识别的标签，而免疫佐剂可以有效增强机体免疫系统对抗原的识别。癌症疫苗发挥作用主要依靠抗原特异性激活的癌症特异性T细胞，即细胞免疫。Toll样受体激动剂是一种可以激活天然免疫反应的物质，是潜在的疫苗佐剂。Toll样受体有很多种，比如Toll样受体3(TLR3)、Toll样受体4、Toll样受体7、Toll样受体8和Toll样受体9(TLR9)。
Poly(I:C)或Poly(ICLC)是Toll样受体3的激动剂，而CpG寡脱氧核苷酸(CpG oligonucleotide,CpG-ODN)是Toll样受体9的激动剂。Poly(I:C)(聚肌苷酸胞苷酸)是一种合成的双链RNA(dsRNA)类似物，是一种与病毒感染相关的分子模式。Poly(I:C)可被TLR3识别，诱导NF-kB的活化和细胞因子的产生。Poly(ICLC)是Poly(I:C)经过适当稳定化修饰的TLR3激动剂，与Poly(I:C)功能相似。CpG-ODN是人工合成的含有非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)的寡脱氧核苷酸(ODN)，可模拟细菌DNA刺激多种哺乳动物包括人的免疫细胞。根据不同的化学结构和生物学特性，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。A类CpG-ODN以含有CpG二核苷酸的回文序列为核心，两端为poly G尾，磷酸二酯键骨架为部分硫代修饰，通过回文序列和poly G形成高级结构，能够活化浆细胞样树突状细胞诱生大量I型干扰素，对B细胞活性弱。B类CpG-ODN是一种全硫代修饰的线性CpG ODN，对B细胞有很强的免疫刺激活性，但不能活化浆细胞样树突状细胞。C类CpG-ODN是一类全硫代修饰的CpG ODN，能通过回文序列形成二聚体，兼具A型和B型CpG-ODN的活性，既能活化浆细胞样树突状细胞，又能活化B细胞。
在抗原激活癌症特异性T细胞的过程中，除了抗原表位被提呈到抗原提呈细胞表面外，还需要第二和第三信号的辅助才能激活癌症特异性T细胞，否则抗原无法有效激活癌症特异性T细胞。而佐剂能辅助激活癌症特异性T细胞激活，更好的发挥疫苗的功能。但目前免疫佐剂的功能仍有待进一步开发，以期更充分发挥对癌症特异性T细胞的激活作用。
发明内容
为解决上述技术问题，本发明提供了一种免疫佐剂组合物，并提供了一种共负载 Poly(I:C)/Poly(ICLC)和CpG-ODN(和氨基酸、多肽、脂类、糖类、蛋白质或无机盐)及抗原组分的预防或治疗癌症的疫苗系统。本发明的免疫佐剂组合物可以更好的增强激活癌症特异性T细胞反应时的功效，更好地辅助疫苗发挥功能。
本发明的第一个目的是提供一种免疫佐剂组合物，该免疫佐剂组合物为至少包括以下组分中的(1)和(2)的组合：(1)Poly(I:C)或Poly(ICLC)，(2)CpG-ODN，其中，CpG-ODN为A类CpG-ODN、B类CpG-ODN和C类CpG-ODN中的至少两种，且至少其中一种为B类CpG-ODN或C类CpG-ODN，(3)氨基酸、多肽、脂类、糖类、蛋白质或无机盐。
进一步地，A类CpG-ODN包括但不限于CpG-ODN 2216、CpG-ODN 1585、CpG-ODN 2336等。
进一步地，B类CpG-ODN包括但不限于CpG-ODN 1018、CpG-ODN 2006、CpG-ODN 1826、CpG-ODN 1668、CpG-ODN 2007、CpG-ODN BW006、CpG-ODN SL01等。
进一步地，C类CpG-ODN包括但不限于CpG-ODN 2395、CpG-ODN SL03、CpG-ODN M362等。
进一步地，免疫佐剂组合物中，CpG-ODN的总质量、Poly(I:C)或Poly(ICLC)的质量与氨基酸、多肽、脂类、糖类、蛋白质或无机盐的质量比为0.05-25:1:0.25-50。
进一步的，氨基酸为带正电的氨基酸，优选为至少包括两种带正电氨基酸的组合，如：精氨酸+组氨酸、精氨酸+赖氨酸、精氨酸+赖氨酸+组氨酸。
进一步的，无机盐为可释放H +或酸性物质以形成质子海绵效应的无机盐，优选为铵盐、碳酸盐、碳酸氢盐、磷酸盐。
本发明的第二个目的是提供一种负载上述免疫佐剂组合物的癌症疫苗，该癌症疫苗包括纳米粒子或微米粒子，以及负载于所述纳米粒子或微米粒子上的抗原组分和免疫佐剂组合物，其中，抗原组分为至少一种与癌症类型相同的多肽，优选地，为来源于癌细胞和/或肿瘤组织的全细胞组分抗原。
进一步地，所述全细胞组分抗原的制备方法包括以下步骤：将癌细胞或肿瘤组织用水或不含溶解剂的溶液裂解，收集可溶部分，为水溶性组分；将不可溶部分用溶解剂溶解后转为可溶的部分为非水溶性组分，所述水溶性组分和非水溶性组分为全细胞组分抗原；其中，溶解剂选自尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐(如十二烷基硫酸钠，SDS)、甘油、蛋白质降解酶、白蛋白、卵磷脂、0.1-2000mg/mL的无机盐、Triton、吐温、DMSO(二甲基亚砜)、乙腈、乙醇、甲醇、DMF(N,N-二甲基甲酰胺)、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷、胆碱、Brij TM-35、Octaethylene glycol monododecyl ether(八甘醇单十二醚)、CHAPS(3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐)、Digitonin(洋地黄皂苷)、lauryldimethylamine oxide(N,N-二甲基烷基-C10-16-胺-N-氧化物)、
Figure PCTCN2022092201-appb-000001
非离子型表面活性剂；
或将癌细胞或肿瘤组织用溶解剂裂解，再将裂解物溶解，得到全细胞组分抗原。
细胞组分中水溶性部分和非水溶性部分囊括了整个细胞的成分和组分。其中与正常细 胞成分相同未突变的蛋白质、多肽和基因因为自身免疫系统发育过程中所产生的免疫耐受不会引起免疫反应；而因为癌症产生的基因、蛋白质和多肽的突变因为没有自身免疫系统发育过程中所产生的免疫耐受因而具有免疫原性且可激活机体针对癌细胞的免疫反应。利用全细胞组分中这些因为疾病突变而产生的具有癌细胞特异性免疫原性的物质即可用于癌症的预防和治疗。本发明通过采用含有溶解剂的溶液将细胞中不溶于纯水或不含溶解剂水溶液的组分转化为在特定溶解溶液中可溶并可被用于制备癌症疫苗，从而提高了癌症疫苗所负载的抗原的全面性和免疫原性。
进一步地，肿瘤组织的裂解包括以下步骤：将肿瘤组织切块后研磨，通过细胞过滤网，加入适量水或不含溶解剂的溶液，反复冻融，并可伴有超声以破坏裂解细胞，待细胞裂解后将裂解物离心，上清液为水溶性组分；在沉淀中加入溶解剂，沉淀中转为可溶的部分为非水溶性组分，以上即为制备癌症疫苗的抗原原料来源。
进一步地，癌细胞的裂解包括以下步骤：将癌细胞系进行培养，离心，弃上清，采用纯水或不含溶解剂的溶液重悬细胞，反复冻融，并可伴有超声以破坏裂解细胞，待细胞裂解后将裂解物离心，上清液为水溶性组分；在沉淀中加入溶解剂，沉淀中转为可溶的部分为非水溶性组分，以上即为制备癌症疫苗的抗原原料来源。
进一步地，所述癌症疫苗上连接具有主动靶向功能的靶头，如甘露糖、甘露聚糖、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等。该靶头可带领癌症疫苗靶向到特定组织或细胞，如树突状细胞、巨噬细胞、B细胞、T细胞、NK细胞、NKT细胞、中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴结、胸腺、脾脏、骨髓等。
进一步地，所述抗原组分和免疫佐剂被包载于纳米粒子或微米粒子的内部，和/或负载于纳米粒子或微米粒子的表面。其中，Poly(I:C)或Poly(ICLC)和多种CpG-ODN可分别负载于纳米粒子或微米粒子的内部和/或表面。
进一步地，纳米粒子的粒径为1nm-1000nm；微米粒子的粒径为1μm-1000μm；采用纳米粒子或微米粒子制备得到的纳米疫苗或微米疫苗表面为电中性、带负电或带正电。
进一步地，癌症疫苗可以按照纳米尺寸粒子和微米尺寸粒子已开发的制备方法制备得到，包括但不仅限于常见的溶剂挥发法、透析法、挤出法、热熔法。在本发明的一些实施方案中，癌症疫苗采用溶剂挥发法中的复乳法制备得到。
进一步地，纳米粒子或微米粒子的制备材料包括但不限于有机合成高分子材料、天然高分子材料或者无机材料。其中，有机合成高分子材料为生物相容或可降解的高分子材料，包括但不限于PLGA(聚乳酸-羟基乙酸共聚物)、PLA(聚乳酸)、PGA(聚乙醇酸)、PEG(聚乙二醇)、PCL(聚己内酯)、Poloxamer(泊洛沙姆)、PVA(聚乙烯醇)、PVP(聚乙烯吡咯烷酮)、PEI(聚乙烯亚胺)、PTMC(聚三亚甲基碳酸酯)、聚酸酐、PDON(聚对二氧六环酮)、PPDO(聚对二氧环己酮)、PMMA(聚甲基丙烯酸甲酯)、PLGA-PEG、PLA-PEG、PGA-PEG、聚氨基酸、合成多肽、合成脂质等；天然高分子材料为为生物相容或可降解的高分子材料，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、淀粉、糖类、多肽等；无机材料为无明显生物毒性的材料，包括但不限于三氧化二铁、四氧化三铁、碳酸钙、磷酸钙等。
本发明的第三个目的是提供上述免疫佐剂组合物或癌症疫苗在制备癌症治疗药物或预防药物中的应用。
本发明的第四个目的是提供上述免疫佐剂组合物在制备细胞免疫激活剂中的应用。B类CpG-ODN对B细胞有很强的免疫刺激活性，但不能活化浆细胞样树突状细胞，C类CpG-ODN既能活化浆细胞样树突状细胞，又能活化B细胞，但是主要活化B细胞。因此，理论上来说，B类和C类CpG-ODN对体液免疫激活效果更好，在细胞免疫激活方面的辅助作用较差；而A类CpG-ODN能够活化浆细胞样树突状细胞诱生大量I型干扰素，对B细胞活性弱，A类激活细胞免疫效果应更好而激活体液免疫能力更差。但是本发明意外的发现将含有B类CpG-ODN或C类CpG-ODN的CpG-ODN混合佐剂与PolyIC或者Poly ICLC联用时，可以取得意想不到的优良效果，且效果显著优于使用A类CpG-ODN。
进一步地，癌症疫苗中至少有一种抗原与所述药物治疗或预防的疾病对应。
借由上述方案，本发明至少具有以下优点：
本发明中Poly(I:C)和不同CpG-ODN所能激活的靶点不同，氨基酸、多肽、脂类、糖类、蛋白质、无机盐等能够协助和增加溶酶体逃逸，在与抗原组分共负载于纳米疫苗或者微米疫苗时，其效果显著优于Poly(I:C)与一种CpG混用或者使用多种CpG。因此本发明的免疫佐剂组合物可以有效增强所负载佐剂辅助抗原激活特异性免疫反应的能力，对疾病的预防和治疗效果有极大的提升，可用于制备预防和治疗癌症的药物。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1为本发明所述抗原组分为全细胞组分时疫苗的制备过程及应用领域示意图；a:水溶性组分和非水溶性组分分别收集和制备纳米疫苗或微米疫苗的示意图；b:采用含有溶解剂的溶解液溶解全细胞组分和制备纳米疫苗或微米疫苗的示意图；
图2-图17为载有水溶性组分和非水溶性组分的纳米尺寸粒子或微米尺寸粒子的结构示意图，在负载多肽时，也可以将多肽分为水溶性多肽和水不溶性多肽两部分并参照全细胞组分负载；其中，1、细胞或组织组分中的水溶性成分；2、细胞或组织组分中的非水溶性成分；3、免疫佐剂；4、纳米粒子或微米粒子；5、纳米粒子中的内核部分；
图18-图28为主动靶向靶头修饰的载有水溶性和非水溶性组分的纳米粒子或微米粒子的结构示意图，在负载多肽时，也可以将多肽分为水溶性多肽和水不溶性多肽两部分并参照全细胞组分负载；其中，1、细胞或组织组分中的水溶性成分；2、细胞或组织组分中的非水溶性成分；3、免疫佐剂；4、纳米粒子或微米粒子；5、纳米粒子中的内核部分；6、 可以靶向特定细胞或者组织的靶头；
图29-51分别为实施例1-23中将CpG-ODN与Poly(I:C)或Poly(ICLC)混合佐剂与癌症细胞或肿瘤组织裂解物共负载到纳米疫苗或微米疫苗用于预防或治疗癌症时小鼠肿瘤生长速度和生存期实验结果；其中，a、纳米疫苗或微米疫苗预防或治疗癌症时的肿瘤生长速度实验结果(n≥8)；b、纳米疫苗或微米疫苗预防或治疗其他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a中肿瘤生长抑制实验的显著性差异采用ANOVA法分析，图b中显著性差异采用Kaplan-Meier和log-rank test分析；***表示与PBS空白对照组相比p＜0.0005，有显著性差异；##代表与空白纳米粒+细胞裂解物对照组相比p＜0.005，有显著性差异；###代表与空白纳米粒+细胞裂解物对照组相比p＜0.0005，有显著性差异。&或$表示与负载CpG和Poly(I:C)或polyICLC特定比例，或者与只负载1类CpG和Poly(I:C)或polyICLC的疫苗组相比p＜0.05，有显著性差异；&&或$$表示与负载CpG和Poly(I:C)或polyICLC特定比例，或者与只负载1类CpG和Poly(I:C)或polyICLC的疫苗组相比p＜0.01，有显著性差异；
Figure PCTCN2022092201-appb-000002
表示与负载CpG和Poly(I:C)或polyICLC特定比例混合佐剂，且只使用一种CpG的疫苗组相比p＜0.01，有显著性差异；τττ与只负载裂解物组分而不负载任何免疫佐剂的对照疫苗组相比p＜0.005，有显著性差异；ρ代表与使用CpG和PolyIC或PolyICLC混合佐剂且CpG:Poly＝1:1的疫苗组相比p＜0.05，有显著性差异；λλ代表与只使用CpG免疫佐剂和裂解物组分的疫苗组相比p＜0.01，有显著性差异；εε代表与只使用PolyIC免疫佐剂和裂解物组分的疫苗组相比p＜0.01，有显著性差异；δδδ代表与使用只负载两种A类CpG和一种C类CpG免疫佐剂的粒子组相比p＜0.005，有显著性差异；βββ代表与负载裂解物组分和精氨酸，而不负载任何佐剂的疫苗组相比p＜0.005，有显著性差异；
Figure PCTCN2022092201-appb-000003
代表与使用两种CpG和PolyIC或PolyICLC混合佐剂并添加甘氨酸的疫苗组相比p＜0.05，有显著性差异；
Figure PCTCN2022092201-appb-000004
代表与只使用一种CpG和PolyIC或PolyICLC混合佐剂并添加增加溶酶体逃逸物质的疫苗组相比p＜0.05，有显著性差异；
Figure PCTCN2022092201-appb-000005
代表与只使用一种CpG和PolyIC或PolyICLC混合佐剂并添加增加溶酶体逃逸物质的疫苗组相比p＜0.01，有显著性差异；★代表与使用CpG和PolyIC或PolyICLC混合佐剂但是不添加任何增加溶酶体逃逸物质的疫苗组相比p＜0.05，有显著性差异；π代表与使用两种A类CpG和PolyIC或PolyICLC作为混合佐剂的疫苗组相比p＜0.05，有显著性差异；ππ代表与使用两种A类CpG和PolyIC或PolyICLC作为混合佐剂的疫苗组相比p＜0.01，有显著性差异；ψ代表与疫苗+CD4 depletion组相比p＜0.05，有显著性差异；ξξξ代表与疫苗+CD8 depletion组相比p＜0.005，有显著性差异；κκκ代表与疫苗+CD8 depletion+CD4 depletion组相比p＜0.005，有显著性差异。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明所述纳米疫苗和/或微米疫苗系统可用于制备预防和/或治疗癌症的疫苗，其制 备过程及应用领域如图1所示。在制备时可裂解细胞或组织后先分别收集水溶性组分和水不溶性组分并分别制备纳米疫苗或微米疫苗；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全细胞组分并制备纳米疫苗或微米疫苗。
本发明所述全细胞组分在裂解前或(和)裂解后既可经过灭活或(和)变性处理后再制备纳米疫苗或微米疫苗，也可不经过任何灭活、酶处理或(和)变性处理直接制备纳米疫苗或微米疫苗。本发明部分实施例中，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、酶处理、或(和)变性处理，或者也可以细胞裂解前和裂解后均做灭活、酶处理或(和)变性处理；本发明部分实施例中灭活或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放射线辐照、高压、冷冻干燥和甲醛等灭活或变性处理方法。
本发明所述的疫苗系统的结构示意图如图2-28所示。图2-图5中纳米粒子或微米粒子表面和内部均含有免疫佐剂；图6-图9中免疫佐剂只分布于纳米粒子或微米粒子的内部；图10-图13中免疫佐剂只在纳米粒子或微米粒子外表面；图14-图17纳米粒子或微米粒子内部和外表面均无免疫增强佐剂；图2，图6，图10和图14中细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；图3，图7，图11和图15中细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时形成了一个内核部分，内核可在制备过程中生成或通过使用聚合物或无机盐等方式形成；图4，图8，图12和图16中细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时形成了多个内核部分，内核可在制备过程中生成或通过使用聚合物或无机盐等方式形成；图5，图9，图13和图17中细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时位于所形成内核的外层；a：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的水溶性成分；b：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的非水溶性成分；c：纳米粒子或微米粒子内部包载的为细胞或组织组分中的非水溶性成分而表面负载的均为细胞或组织组分中的水溶性成分；d：纳米粒子或微米粒子内部包载的为细胞或组织组分中的水溶性成分而表面负载的均为细胞或组织组分中的非水溶性成分；e：纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面也同时负载细胞或组织组分中的水溶性成分和非水溶性成分；f:纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的水溶性成分；g:纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的非水溶性成分；h：纳米粒子或微米粒子内部只包载的细胞或组织组分中的非水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；i:纳米粒子或微米粒子内部只包载的细胞或组织组分中的水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；
图18-图19中纳米粒子或微米粒子表面和内部均含有免疫佐剂；图20-图21中免疫佐剂只分布于纳米粒子或微米粒子的内部；图22-图23中纳米粒子或微米粒子只在外表面含有免疫佐剂；图24-图25纳米粒子或微米粒子内部和外表面均无免疫佐剂；图26细胞组分和/或免疫佐剂只分布于纳米粒子或微米粒子内部；图27细胞组分和/或免疫佐剂只分布于纳米粒子或微米粒子外部；图28细胞组分和免疫佐剂分别分布于纳米粒子或微米粒子内部或外部。在图18-25中，图18中2.a-2.i，图20中6.a-6.i，图22中10.a-10.i和图24中14.a-14.i纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；图19中3.a-3.i，图20中7.a-7.i，图22中11.a-11.i和图24中15.a-15.i中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的一个内核部分；图19中4.a-4.i，图21中8.a-8.i，图23中12.a-12.i和图25中16.a-16.i纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的多个内核部分；图19中5.a-5.i，图21中9.a-9.i，图23中13.a-13.i和图25中17.a-17.i纳米粒子或微米粒子所包载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部所形成内核的外层；a：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的水溶性成分；b：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的非水溶性成分；c：纳米粒子或微米粒子内部包载的为细胞或组织组分中的非水溶性成分而表面负载的均为细胞或组织组分中的水溶性成分；d：纳米粒子或微米粒子内部包载的为细胞或组织组分中的水溶性成分而表面负载的均为细胞或组织组分中的非水溶性成分；e：纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面也同时负载细胞或组织组分中的水溶性成分和非水溶性成分；f:纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的水溶性成分；g:纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的非水溶性成分；h：纳米粒子或微米粒子内部只包载的细胞或组织组分中的非水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；i:纳米粒子或微米粒子内部只包载的细胞或组织组分中的水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分。在图26-28中，a,b和c中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；d,e和f中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的一个内核部分；g，h和i中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的多个内核部分；j，k和l中纳米粒子或微米粒子所包载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部所形成内核的外层；a,d,g 和j中纳米粒子或微米粒子负载的均为细胞或组织组分中的水溶性成分；b，e，h和k中纳米粒子或微米粒子负载的均为细胞或组织组分中的非水溶性成分；c，f，i和l中纳米粒子或微米粒子同时负载细胞或组织组分中的水溶性成分和非水溶性成分。
在实际使用过程中可以只使用其中某一种特定结构的纳米粒子和/或微米粒子，或者是同时使用一种或多种以上的不同结构的纳米粒子和/或微米粒子。
在一些实施方案中，本发明所采用的复乳法的具体制备方法如下：
步骤1，将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度医用材料的有机相中。
在一些实施例中，水相溶液可含有癌细胞裂解物中的各组分以及免疫佐剂；癌细胞裂解物中的各组分在制备时分别为水溶性组分或者是溶于8M尿素或6M盐酸胍等溶解剂中的原非水溶性组分。水相溶液所含有来自癌细胞的水溶性组分的浓度或者是来自癌细胞的溶于溶解剂的原非水溶性组分的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL，能负载足够癌症抗原以激活相关免疫反应。免疫增强佐剂在初始水相中的浓度为大于0.01ng/mL。
医用高分子材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL，优选为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，在本发明中，水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000，优选为1:10。
优选的，水相溶液为裂解物组分溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL；水相溶液为裂解物组分/免疫佐剂溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL，免疫佐剂的浓度大于0.01ng/mL，优选0.01mg/mL～20mg/mL。高分子材料有机相溶液中，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；高分子材料的浓度为0.5mg/mL～5000mg/mL，优选为100mg/mL。
步骤2，将步骤1得到的混合液进行超声处理、搅拌、均质处理或微流控处理。优选的，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于1分钟，比如搅拌速度为50rpm～1500rpm，搅拌时间为0.1小时～24小时；超声处理时，超声功率大于5W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于5psi，比如20psi～100psi，使用高剪切均质机时转速大于100rpm，比如1000rpm～5000rpm；使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米和/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大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。 该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。该步骤是为了进行纳米化或微米化，超声时间长短或搅拌速度及时间能控制制备的纳米粒子或微米粒子大小，过长或过短都会带来粒径大小的变化，为此，需要选择合适的超声时间。在本发明中，超声时间大于0.1秒，比如2～200秒，搅拌速度大于50rpm，比如50rpm～500rpm，搅拌时间大于1分钟，比如60～6000秒。优选的，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。
在本发明中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中，第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定，优选为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。同样地，本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。
本步骤中，乳化剂水溶液依然为PVA。
第四预定浓度为5mg/mL，第四预定浓度的选择，以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中，第三预定体积与第三预定体积之比为范围为1:1.5-1:2000，优选为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中，本步骤的预定搅拌条件为直至有机溶剂挥发完成，也即步骤1中的二氯甲烷挥发完成。
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于100rpm的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
在本发明一些实施方案中，步骤5所得沉淀重新混悬于第六预定体积的PBS(或生理盐水)中时不需要冻干，可直接进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验或者直接作为纳米疫苗或微米疫苗使用。
在本发明一些实施方案中，步骤5所得沉淀重新混悬于含有冻干保护剂的水溶液中时需进行冷冻干燥，再冷冻干燥以后再进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物 的相关实验或者直接作为纳米疫苗或微米疫苗使用。
在本发明中，所述冻干保护剂选用海藻糖(Trehalose)。
在本发明中，该步骤的冻干保护剂的第五预定浓度为质量百分比4％，之所以如此设定，是为了在后续进行冷冻干燥中不影响冻干效果。
步骤6，将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后，将冻干物质备用。
步骤7，将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，与第七预定体积的水溶性组分或者溶于8M尿素等溶解剂中的原非水溶性组分混合后即得纳米疫苗或微米疫苗。
在本发明中，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。
在一些实施例中，所述重悬的纳米粒子混悬液体积为9mL时，含有癌细胞裂解物或含有肿瘤组织裂解物中的水溶性组分或者溶于溶解剂中的原非水溶性组分的体积与为1mL。在实际使用时可将二者体积和比例根据需要进行调整。
纳米疫苗或微米疫苗的粒径大小为纳米级或微米级，这样能保证疫苗被抗原提呈细胞吞噬，而为了提高吞噬效率，粒径大小要在适宜的范围内。纳米疫苗的粒径大小为1nm-1000nm，更优选地，粒径大小为30nm-1000nm，最优选地，粒径大小为100nm-600nm；微米疫苗的粒径大小为1μm-1000μm，更优选地，粒径大小为1μm-100μm，更优选地，粒径大小为1μm-10μm，最优选地，粒径大小为1μm-5μm。本实施例中，纳米粒疫苗粒径大小为100nm-600nm，微米疫苗粒径大小为1μm-5μm。
在另一些实施方案中，采用复乳法制备纳米疫苗或微米疫苗的具体方法如下：
步骤1，将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度医用材料的有机相中。
在一些实施例中，水相溶液可含有癌细胞裂解物中的各组分以及免疫增强佐剂poly(I:C)和CpG-ODN，或者Poly ICLC和CpG-ODN；水相溶液所含有来自癌细胞的水溶性组分的浓度或者是来自癌细胞的溶于溶解剂的原非水溶性组分的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL，能负载足够癌症抗原以激活相关免疫反应。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，医用材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL，优选为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，在本发明中，水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000，优先地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒或微米粒的尺寸大小。
优选的，水相溶液为裂解物组分溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL；水相溶液为裂解物组分/免疫佐剂溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL，免疫佐剂的浓度大于0.01ng/mL，优选0.01mg/mL～20mg/mL。高分子材料有机相溶液中，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；高分子材料的浓度为0.5mg/mL～5000mg/mL，优选为100mg/mL。
步骤2，将步骤1得到的混合液进行超声处理、搅拌、均质处理或微流控处理。优选的，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于1分钟，比如搅拌速度为50rpm～1500rpm，搅拌时间为0.1小时～24小时；超声处理时，超声功率大于5W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于5psi，比如20psi～100psi，使用高剪切均质机时转速大于100rpm，比如1000rpm～5000rpm；使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的微纳粒子大小，过大或过小都会带来粒径大小的变化。
步骤3，将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行超声处理、搅拌、均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。该步骤是为了进行纳米化或微米化，超声时间长短或搅拌速度及时间能控制制备的纳米粒子或微米粒子大小，过长或过短都会带来粒径大小的变化，为此，需要选择合适的超声时间。在本发明中，超声时间大于0.1秒，比如2～200秒，搅拌速度大于50rpm，比如50rpm～500rpm，搅拌时间大于1分钟，比如60～6000秒。优选的，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。
在本发明中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中，第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定，优选为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。同样地，本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件或者也可不进行搅拌直接进行后续处理。
本步骤中，乳化剂水溶液依然为PVA。
第四预定浓度为5mg/mL，第四预定浓度的选择，以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中，第三预定体积与第三预定体积之比为范围为1:1.5-1:2000，优先地为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中，本步骤的预定搅拌条件为有机溶剂挥发完成，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全细胞组分中水溶性和/或非水溶性组分的溶液中，或者将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有全细胞组分中水溶性和/或非水溶性组分与佐剂混合的溶液中。
步骤6，将步骤5处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第六预定体积的固化处理试剂或矿化处理试剂，作用一定时间后离心洗涤，然后加入第七预定提交的含有带正电或者带负电的物质并作用一定时间。
在本发明一些实施方案中，步骤6所得沉淀重新混悬于第七预定体积的带电物质后可不需要冻干，可直接进行后续纳米粒子或微米粒子表面负载癌细胞/组织裂解物的相关实验，或者直接作为纳米疫苗或微米疫苗使用。
在本发明一些实施方案中，步骤6所得沉淀重新混悬于含有干燥保护剂的水溶液中后进行室温真空干燥或者冷冻真空干燥，在干燥以后再进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验，或者直接作为纳米疫苗或微米疫苗使用。
在本发明中，所述冻干保护剂选用海藻糖(Trehalose)，或者甘露醇与蔗糖的混合溶液。在本发明中，该步骤的干燥保护剂的浓度为质量百分比4％，之所以如此设定，是为了在后续进行干燥中不影响干燥效果。
步骤7，将步骤6得到的含有干燥保护剂的混悬液进行干燥处理后，将干燥后的物质备用。
步骤8，将第八预定体积的步骤6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八预定体积的PBS(或生理盐水)重悬步骤7得到的干燥后的含有纳米粒或微米粒和干燥保护剂的干燥后物质直接使用；或者与第九预定体积的水溶性组分或者非水溶性组分混合后使用。
在本发明中，步骤5-步骤8的修饰和抗原负载步骤可重复多次以提高抗原的负载量。而且在添加带正电或带负电的物质时可以多次添加带同种电荷的或者也可以交替添加带不同电荷的物质。
在一些实施例中，所述重悬的纳米粒子混悬液体积为9mL时，含有癌细胞裂解物或含有肿瘤组织裂解物中的水溶性组分或者原非水溶性组分的体积与为0.1-100mL。在实际使用时可将二者体积和比例根据需要进行调整。
实施例1佐剂和黑色素瘤肿瘤组织抗原负载于纳米粒子内部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制备共负载免疫佐剂和黑色素瘤肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗预防黑色素瘤。本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。首先裂解B16F10黑色素瘤肿瘤组织并制备肿瘤组织的水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以CpG-ODN 1018(B类)、CpG-ODN 2395(C类)和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组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来预防黑色素瘤。
(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1.5×10 5个B16-F10细胞，在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨，通过细胞过滤网加入适量纯水并反复冻融5次，并可伴有超声以破坏裂解组织细胞。待组织细胞裂解后，将裂解物以5000g的转速离心3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8M尿素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8M尿素水溶液中可溶。
(2)纳米疫苗的制备
本实施例中纳米疫苗及作为对照的空白纳米粒采用溶剂挥发法制备，所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa，负载水溶性组分的纳米疫苗和负载非水溶性组分的纳米疫苗分别制备，使用时一起使用。采用的免疫佐剂为CpG-ODN 1018、CpG-ODN 2395和poly(I:C)，三者之间的质量比为0.5：1：1。制备方法如前所述，PLGA纳米粒子将免疫佐剂和全细胞组分包载于纳米疫苗中。负载全细胞组分的纳米疫苗平均粒径为280nm左右，纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的佐剂总质量为0.05mg，其中CpG-ODN 1018为0.01mg，CpG-ODN 2395为0.02mg，poly(I:C)为0.02mg。只负载0.05mg免疫佐剂(CpG-ODN 1018为0.01mg，CpG-ODN 2395为0.02mg，poly(I:C)为0.02mg)的空白纳米粒粒径为270nm左右，空白纳米粒制备时分别采用含有等量佐剂的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。以来自B16F10肿瘤组织的水溶性组分和溶解于8M尿素中的原非水溶性组分为原料来源制备只负载B16F10肿瘤组织全细胞组分的疫苗，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分，纳米疫苗表面电位为-5mV左右。对照纳米疫苗粒径为280nm，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的佐剂总质量为0.05mg，其中，CpG-ODN 1018为0.0125mg、CpG-ODN 2395为0.0125mg，poly(I:C)为0.025mg，纳米疫苗表面电位为-5mV左右。
(3)纳米疫苗用于癌症的预防
本研究对照组分别是PBS组和空白纳米粒+游离组织裂解物组。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。
纳米疫苗组给药方案如下：在接种黑色素瘤之前第42天、第35天、第28天、第21天、和第7天分别皮下注射100μL负载水溶性成分的1mg PLGA纳米疫苗和100μL负载原非水溶性成分的1mg PLGA纳米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
PBS对照组方案如下：在接种黑色素瘤之前第42天、第35天、第28天、第21天、和第7天分别皮下注射200μL PBS；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
空白纳米粒+游离裂解物对照组：在接种黑色素瘤之前第42天、35天、28天、21天、7天分别皮下注射200μL空白纳米粒和与等量的游离裂解物；空白纳米粒和游离裂解物注射在不同部位；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图29所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大且生产速度很快。纳米疫苗组的疗效都好于PBS对照组和空白纳米粒对照组，而且负载1.5:1(CpG-ODN：poly(I:C))比例混合佐剂和肿瘤组织全细胞组分的纳米疫苗处理组小鼠的肿瘤在接种后大部分消失，好于负载1:1(CpG-ODN：poly(I:C))比例混合佐剂和肿瘤组织全细胞组分的疫苗。综上所述，本发明所述的负载特定比例的多种CpG-ODN和Poly(I:C)及抗原组分的纳米疫苗对黑色素瘤具有良好的预防效果。
实施例2佐剂和多肽负载于纳米粒子内部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制备共负载免疫佐剂和多肽的纳米疫苗，并应用该疫苗预防黑色素瘤。本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。以有机高分子材料PLGA为纳米粒骨架材料，以poly(I:C)和CpG-ODN 2006(B类)、CpG-ODN 2216(A类)为免疫佐剂采用溶剂挥发法制备负载佐剂和多肽抗原的纳米疫苗。然后采用该纳米疫苗来预防黑色素瘤。
(1)多肽抗原
本实施例采用水溶性多肽抗原B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M27(REGVELCPGNKYEMRRHGTTHSLVIHD)和8M尿素水溶液(含500mM氯化钠)溶解的水不溶性多肽抗原B16-M05(Eef2,FVVKAYLPVNESFAFTADLRSNTGGQA),B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ),and TRP2:180-188(SVYDFFVWL)。将水 溶性多肽抗原和8M尿素水溶液(含500mM氯化钠)溶解的水不溶性多肽抗原中所含各多肽抗原按照质量比等量混合作为抗原组分使用。
(2)纳米疫苗的制备
本实施例中纳米疫苗及作为对照的空白纳米粒以及负载多种多肽的纳米粒采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫佐剂为CpG-ODN 2006(B类)、CpG-ODN 2216(A类)和poly(I:C)，三者质量比为1:1:0.5。制备方法如前所述。负载多肽的纳米疫苗平均粒径为270nm左右，纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载70μg多肽组分，每1mgPLGA纳米粒内外所使用的免疫佐剂共0.05mg，其中CpG-ODN 2006为0.02mg，CpG-ODN 2216为0.02mg，poly(I:C)为0.01mg。只含免疫佐剂空白纳米粒粒径为250nm左右，空白纳米粒制备时分别采用含有等量佐剂的纯水或8M尿素水溶液(含500mM氯化钠)代替相对应的多肽。对照多肽纳米疫苗粒径约为270nm,每1mg PLGA纳米粒子约负载70μg多肽组分，每1mgPLGA纳米粒内外所使用的免疫佐剂共0.05mg，其中CpG-ODN 2006为0.025mg、CpG-ODN 2216为0.024mg，poly(I:C)为0.001mg。
(3)纳米疫苗用于癌症的预防
本研究对照组分别是PBS组和空白纳米粒+游离多肽组。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。
纳米疫苗组给药方案如下：在接种黑色素瘤之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL的2mg PLGA纳米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
PBS对照组方案如下：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射200μL PBS；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
空白纳米粒+游离多肽对照组：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射200μL空白纳米粒和与疫苗负载的等量的游离裂解物；空白纳米粒和游离多肽注射在不同部位；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图30所示，PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大且生产速度很快。纳米疫苗组的疗效都好于PBS对照组和空白纳米粒对照组，而且负载4:1(CpG-ODN：poly(I:C))比例混合佐剂和多肽抗原的纳米疫苗处理组小鼠的肿瘤在接种后大部分消失，好于负载49:1(CpG-ODN：poly(I:C))比例混合佐剂和多肽抗原的疫苗。综上所述，本发明所述的负载特定比例的多种CpG和Poly(I:C)及抗原组分的纳米疫苗对黑色素瘤具有 良好的预防效果。
实施例3佐剂和黑色素瘤癌细胞负载于纳米粒子内部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制备共负载免疫佐剂和黑色素瘤癌细胞全细胞组分的纳米疫苗，并应用该疫苗治疗黑色素瘤。本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。首先裂解B16F10黑色素瘤癌细胞并制备水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以poly(I:C)、CpG-ODN 1018(B类)、CpG-ODN 1826(B类)和CpG-ODN 2336(A类)为免疫佐剂采用溶剂挥发法制备负载有癌细胞的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗黑色素瘤。
(1)细胞的裂解及各组分的收集
将培养的B16F10癌细胞系去除培养基后在400g离心5分钟，使用PBS重悬并洗涤两遍，然后使用超纯水重悬癌细胞，并反复冻融五次，冻融过程中使用超声辅助裂解癌细胞，然后在10000g离心5分钟，收集上清液即为水溶性组分。将沉淀部分使用8M尿素溶解，即为溶解的原非水溶性组分。将来自B16F10癌细胞的水溶性组分和溶解于8M尿素中的原非水溶性组分按照质量比1:1的比例混合即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa，佐剂和抗原组分包载于纳米疫苗中，所采用的免疫佐剂为poly(I:C)、CpG-ODN 1018、CpG-ODN 1826和CpG-ODN 2336。制备方法如前所述。负载全细胞组分的纳米疫苗平均粒径为320nm左右；纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的佐剂为0.05mg，其中poly(I:C)为0.01mg，CpG-ODN 1018为0.01mg，CpG-ODN 1826为0.01mg，CpG-ODN 2336为0.02mg。对照纳米疫苗的制备方法相同，粒径为320nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mgPLGA纳米粒使用佐剂为0.05mg，poly(I:C)为0.049mg，CpG-ODN 1018为0.0003mg，CpG-ODN 1826为0.0003mg，CpG-ODN 2336为0.0004mg。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。
纳米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞；在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射100μL的2mg PLGA纳米疫苗。
PBS对照组方案如下：在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞；在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射200μL 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×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图31所示，PBS对照组小鼠的肿瘤都长大且生产速度很快。纳米疫苗组的疗效都好于PBS对照组，而且负载0.5:0.5:1:0.5(CpG-ODN 1018：CpG-ODN 1826：CpG-ODN 2336：poly(I:C))比例混合佐剂和肿瘤组织全细胞组分的纳米疫苗处理组小鼠的肿瘤在接种后大部分消失，好于负载3:3:4:490(CpG-ODN 1018：CpG-ODN 1826：CpG-ODN 2336：poly(I:C))比例混合佐剂和肿瘤组织全细胞组分的疫苗。综上所述，本发明所述的负载特定比例的多种CpG和Poly(I:C)及抗原组分的纳米疫苗对黑色素瘤具有良好的治疗效果。
实施例4佐剂和黑色素瘤肿瘤组织负载于纳米粒子内部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制备共负载免疫佐剂和黑色素瘤肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗治疗黑色素瘤。本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。首先裂解B16F10黑色素瘤肿瘤组织并制备水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以poly(I:C)、CpG-ODN 2336(A类)和CpG-ODN 1018(B类)为免疫佐剂采用溶剂挥发法制备负载有癌细胞的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗黑色素瘤。
(1)肿瘤组织的裂解及各组分的收集
同实施例1。将水溶性组分和溶解于5％脱氧胆酸钠中的原非水溶性组分按照质量比1:1的比例混合即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗采用复乳法制备，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，佐剂和抗原组分包载于纳米疫苗中，所采用的免疫佐剂为poly(I:C)、CpG-ODN 2336和CpG-ODN 1018。制备方法如前所述。负载全细胞组分的纳米疫苗平均粒径为350nm左右；纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的佐剂为0.03mg，其中poly(I:C)为0.01mg，CpG-ODN 2336为0.01mg，CpG-ODN 1018为0.01mg。对照纳米疫苗1的制备方法相同，粒径为340nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mg PLGA纳米粒使用0.03mg佐剂，其中0.01mg poly(I:C)、0.01mg CpG-ODN 2336和0.01mg CpG-ODN 2216。对照纳米疫苗2的制备方法相同，粒径为340nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mg PLGA纳米粒使用0.03mg佐剂，其中0.01mg CpG-ODN 1018、0.01mg CpG-ODN 2336和0.01mg CpG-ODN 2216；
(3)纳米疫苗用于癌症的治疗
同实施例3。
(4)实验结果
如图32所示，纳米疫苗组的疗效都好于PBS对照组，而且负载CpG-ODN(B类)：CpG-ODN(A类)：poly(I:C)＝1:1:1比例混合佐剂和肿瘤组织全细胞组分的纳米疫苗处理组小鼠的肿瘤在接种后大部分消失，好于负载CpG-ODN(A类)：CpG-ODN(A类)：poly(I:C)＝1:1:1比例混合佐剂和肿瘤组织全细胞组分的对照纳米疫苗1处理组小鼠，也好于负载CpG-ODN(A类)：CpG-ODN(A类)：CpG-ODN(B类)＝1:1:1比例混合佐剂和肿瘤组织全细胞组分的对照纳米疫苗2处理组小鼠。综上所述，本发明所述的负载特定比例的多种CpG和Poly(I:C)及抗原组分的纳米疫苗对黑色素瘤具有良好的治疗效果。
实施例5黑色素瘤和肺癌细胞水溶性细胞组分负载于微米粒子内部和表面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制备只负载有黑色素瘤和肺癌细胞组分中水溶性部分的微米疫苗，并应用该疫苗预防黑色素瘤。本实施例中，首先裂解B16F10黑色素瘤和LLC肺癌细胞以制备水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA(24KDa-38KDa)为微米粒子骨架材料，以poly(I:C)和两种CpG-ODN为免疫佐剂制备负载有全细胞的水溶性组分的微米疫苗，并应用该微米疫苗预防黑色素瘤。
(1)癌细胞的裂解及各组分的收集
收集一定量的B16F10细胞或LLC细胞，去除培养基后采用-20℃冷冻，加一定量超纯水后通过加热和紫外照射处理细胞，然后反复冻融3次以上，并伴有超声破坏裂解细胞。待细胞裂解后，将裂解物以8000g的转速离心5min取上清液即为B16F10黑色素瘤或LLC肺癌细胞中可溶于纯水的水溶性组分。上述所得来源于两种癌细胞裂解物的水溶性组分按质量比1:1混合即为制备微米疫苗的抗原来源。
(2)微米疫苗的制备
本实施例中制备微米疫苗及作为对照的空白微米粒采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料为PLGA，所采用的免疫佐剂为poly(IC)、CpG-ODN 2006(B类)和CpG-ODN2216(A类)，三者质量比为2:2:1。抗原组分包载于微米疫苗内部和吸附于微米粒子表面，佐剂只包载于微米疫苗内部。制备方法如前所述。在微米粒子表面负载细胞组分和免疫佐剂后所得微米疫苗粒径为2.10μm，表面电位为-5mV左右，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用佐剂0.05mg，其中Poly(I:C)0.02mg，CpG-ODN 2006 0.02mg，CpG-ODN2216 0.01mg。对照微米疫苗1只使用一种CpG和poly(I:C)作为混合佐剂，粒径为2.10μm，表面电位为-5mV，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用佐剂0.05mg，其中Poly(IC)0.02mg，CpG-ODN2006 0.03mg。对照微米疫苗2粒径为2.10μm，表面电位为-5mV，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用佐剂0.05mg，其中Poly(IC)0.025mg，CpG-ODN 2006 0.02mg，CpG-ODN 2216 0.005mg。
(3)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备黑色素瘤荷瘤小鼠。
微米疫苗组方案如下：在接种黑色素瘤之前第35天、第28天、第21天、第14天分别皮下注射200μL负载癌细胞裂解物中水溶性成分的2mg PLGA微米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。PBS空白对照组方案如下：在接种黑色素瘤之前第35天、第28天、第21天、第14天分别皮下注射200μL PBS；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。由于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图33所示，与PBS空白对照组相比，微米疫苗给药组中小鼠肿瘤体积生长速度均明显变慢且小鼠生存期均明显延长。而且，使用CpG-ODN：Poly(I:C)＝1.5:1作为佐剂的微米疫苗好于使用CpG-ODN:Poly(I:C)比例为1:1作为佐剂的微米疫苗组，而且，使用两种CpG效果优于只使用一种CpG。
实施例6肝癌肿瘤组织裂解组分负载于微米粒子内部和表面用于肝癌的预防
本实施例制备负载有肝癌肿瘤组织裂解物组分的微米疫苗，并应用该疫苗预防肝癌。本实施例中，将小鼠肝癌肿瘤组织裂解组分负载于微米粒子内部和表面以制备微米疫苗。首先取得小鼠肝癌肿瘤组织，使用8M尿素裂解肿瘤组织并溶解肿瘤组织裂解物组分。然后，以PLGA(38KDa-54KDa)为微米粒骨架材料，以Poly ICLC、CpG-ODN M362(C类)和CpG-ODN2216(A类)为免疫佐剂制备微米疫苗,并应用该微米疫苗预防Hepa 1-6肝癌荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种2×10 6个Hepa 1-6细胞或者2×10 6个LLC肺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切碎过细胞筛网，然后使用8M尿素裂解肿瘤组织后溶解。
(2)微米疫苗的制备
本实施例中制备微米疫苗及作为对照的空白微米粒采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料为PLGA，所采用的免疫佐剂为Poly ICLC、CpG-ODN M362和CpG-ODN 2216，三者质量比为1:3:1。抗原组分包载于微米疫苗内部和吸附于微米粒子表面，佐剂只包载于微米疫苗内部。制备时先采用溶解挥发法将抗原组分和佐剂包载于微米粒子内部，然后在8000g离心15min,收集沉淀并将100mg PLGA微米粒子使用4％海藻糖溶液重悬，然后冷冻干燥48小时后冷藏备用。在注射使用前，将10mg PLGA微米粒子使用0.9mL PBS或生理盐水重悬，然后与0.1mL含有8M尿素中的细胞裂解液(60mg/mL)和免疫佐剂(2mg/mL,其中质量比Poly ICLC:CpG-ODN M362：CpG-ODN2216＝1:3:1)的样品在室温混合作用10min,即可注射使用。所得微米疫苗粒径为2.10μm，表面电位为-5mV左右，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用佐剂0.05mg，其中Poly  ICLC 0.01mg，CpG-ODN M362 0.03mg，CpG-ODN 2216 0.01mg。对照微米疫苗粒径为2.10μm，表面电位为-5mV，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用Poly ICLC、CpG-ODN M362和CpG-ODN 2216作为佐剂，其中Poly ICLC 0.025mg，CpG-ODN M362 0.0125mg，CpG-ODN 2216 0.0125mg。只负载免疫佐剂的空白微米粒粒径为2.00μm，空白微米粒制备时分别采用含有等量Poly ICLC，CpG-ODN M362和CpG-ODN 2216的8M尿素代替相对应的裂解物组分。
(3)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备Hepa 1-6肝癌荷瘤小鼠。
在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL的2mg PLGA微米疫苗。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。
PBS空白对照组方案如下：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL PBS。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。
空白微米粒+游离裂解物对照组：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL空白微米粒和与疫苗所负载的等量的游离裂解物；空白微米粒和游离细胞裂解物注射在不同部位。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图34所示，PBS对照组以及空白微米粒+游离裂解物对照组小鼠的肝癌肿瘤生长均较快。微米疫苗给药组小鼠在接种肿瘤后大部分小鼠肿瘤消失。由此可见，本发明所述的负载肝癌肿瘤组织裂解物的微米疫苗对肝癌具有预防效果。而且，佐剂中多种CpG-ODN总含量：Poly ICLC＝4:1的微米疫苗效果好于佐剂中多种CpG-ODN总含量：Poly ICLC＝1:1的微米疫苗。
实施例7肺癌和肝癌肿瘤组织全细胞组分负载于纳米粒子内部用于肝癌的预防
本实施例以小鼠肝癌为癌症模型来说明如何制备负载有肺癌和肝癌肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗预防肝癌。首先裂解肺癌和肝癌肿瘤组织以制备全细胞组分的水溶性组分和非水溶性组分，并将水溶性组分按质量比1:1的比例混合，将非水溶性组分也按质量比1:1的比例混合。然后，以PLGA为纳米粒子骨架材料，以Poly(I:C)、CpG-ODN 1826(B类)和CpG-ODN 1018(B类)按质量比1:1:1混合作为免疫佐剂制备负载有水溶性组分或负载有非水溶性组分的纳米疫苗，使用时将负载水溶性组分的纳米疫苗和负载非水溶性组分的纳米疫苗混合使用预防肝癌。
(1)癌细胞的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种1.0×10 6个LLC细胞或者1.0×10 6个Hepa 1-6细胞，在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨， 通过细胞过滤网加入适量纯水并反复冻融5次，并伴有超声以破坏裂解组织细胞。待组织细胞裂解后，使用核酸酶降解全细胞组分中的核酸，然后在95℃加热5分钟灭活核酸酶，尔后将裂解物以1000g的转速离心5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％辛基葡萄糖苷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％辛基葡萄糖苷水溶液中可溶。收集到水溶性组分和非水溶性组分后，将水溶性组分按质量比1:1的比例混合，将非水溶性组分也按质量比1:1的比例混合。所得水溶性组分混合物或非水溶性组分混合物即为制备纳米疫苗的抗原原料。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，以Poly(I:C)、CpG-ODN 1826和CpG-ODN 1018按质量比1:1:1作为混合佐剂使用。在制备疫苗时，水溶性组分混合物和非水溶性组分混合物分别制备成纳米疫苗再一起使用，抗原组分和佐剂只采用纳米粒包载的方式负载于纳米疫苗。制备方法同上。纳米疫苗粒径为280nm左右，表面电位为-6mV左右，每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.03mg。空白纳米粒粒径为250nm左右，空白纳米粒制备时分别采用含有等量佐剂的溶液代替相对应的水溶性组分和非水溶性组分。对照纳米疫苗粒径为280nm左右，表面电位为-6mV左右，每1mg PLGA纳米粒子约负载100μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂Poly(I:C)共0.03mg或者CpG-ODN 1826+CpG-ODN 1018共0.03mg(质量比1:1)。
(3)纳米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备Hepa 1-6肝癌荷瘤小鼠。在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射100μL的1mg PLGA水溶性组分纳米疫苗和100μL的1mg PLGA非水溶性组分纳米疫苗。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。PBS空白对照组方案如下：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL PBS。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。空白纳米粒+游离裂解物对照组：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL空白纳米粒和与疫苗所负载的等量的游离裂解物；空白纳米粒和游离细胞裂解物注射在不同部位。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图35所示，与对照组相比，疫苗预防组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且，同时使用CpG-ODN和Poly(I:C)(总CpG-ODN与Poly(I:C)质量比2:1)作为佐剂的纳米疫苗预防效果好于只使用Poly(I:C)或者只使用CpG-ODN的纳米疫苗。
实施例8胰腺癌肿瘤组织裂解组分负载于纳米粒子内部用于胰腺癌的治疗
本实施例以小鼠胰腺癌为癌症模型来说明如何制备负载有胰腺癌肿瘤组织裂解物组分的纳米疫苗，并应用该疫苗治疗胰腺癌。首先取得小鼠胰腺癌肿瘤组织并将其裂解以制备水溶性组分和溶于6M盐酸胍中的原非水溶性组分。以PLGA(分子量7KDa-17KDa)为纳米粒子骨架材料，以Poly(I:C)、CpG-ODN 2395和CpG-ODN 2216为免疫佐剂制备纳米疫苗。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种1×10 6个Pan02胰腺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。肿瘤组织的裂解方法及各组分的收集方法同上，只是使用6M盐酸胍代替8M尿素。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，以Poly(I:C)：CpG-ODN 2395：CpG-ODN 2216按质量比1:0.6:0.6作为混合佐剂使用。在制备疫苗时，水溶性组分混合物和非水溶性组分混合物分别制备成纳米疫苗再一起使用，抗原组分和佐剂只采用纳米粒包载的方式负载于纳米疫苗。制备方法同上。纳米疫苗粒径为270nm左右，表面电位为-5mV左右，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.044mg，其中Poly(I:C)为0.02mg，CpG-ODN 2395为0.012mg：CpG-ODN 2216为0.012mg。对照纳米疫苗1粒径为280nm左右，表面电位为-5mV左右，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.044mg,其中Poly(I:C)为0.02mg，CpG-ODN 1585为0.012mg：CpG-ODN 2216为0.012mg。对照纳米疫苗2粒径为280nm左右，表面电位为-6mV左右，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.044mg,其中CpG1585为0.02mg，CpG-ODN 2395为0.012mg：CpG-ODN 2216为0.012mg。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6制备胰腺癌瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1×10 6个个Pan02细胞。疫苗组在第4天、第7天、第10天、第15天和第20天分别皮下注射100μL负载水溶性成分的1mg PLGA纳米疫苗和100μL负载非水溶性成分的1mg PLGA纳米疫苗。PBS空白对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。在实验中，小鼠肿瘤监测和体积计算方法同上。
(4)实验结果
如图36所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且负载CpG-ODN(C类)：CpG-ODN(A类)：poly(I:C)＝0.6:0.6:1比例混合佐剂和肿瘤组织全细胞组分的纳米疫苗处理组小鼠的肿瘤在接种后大部分消失，好于负载CpG-ODN(A类)：CpG-ODN(A类)：poly(I:C)＝0.6:0.6:1比例混合佐剂和肿瘤组织全细胞组分的对照纳米疫苗1处理组小鼠。也 好于负载CpG-ODN(A类)：CpG-ODN(C类)：CpG-ODN(A类)：＝0.6:0.6:1比例混合佐剂和肿瘤组织全细胞组分的对照纳米疫苗2处理组小鼠；综上所述，本发明所述的负载特定比例的多种CpG和Poly(I:C)及抗原组分的纳米疫苗对癌症具有良好的治疗效果。
实施例9全细胞组分负载于甘露糖修饰的微米粒子内部用于乳腺癌的预防
本实施例以小鼠乳腺癌为癌症模型来说明如何制备负载有乳腺癌肿瘤组织和癌细胞的全细胞组分的微米疫苗，并应用该疫苗预防乳腺癌。在实际应用时粒子大小，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，首先取得小鼠乳腺癌和肺癌的癌细胞并将其裂解以制备水溶性组分和溶于8M尿素中的原非水溶性组分。然后，以PLGA和甘露糖修饰的PLGA为微米粒骨架材料，以Poly(I:C)、CpG-ODN 1018(B类)和CpG-ODN 1826(B类)为免疫佐剂采用溶剂挥发法制备微米疫苗。该微米疫苗具有靶向树突状细胞的能力。
(1)肿瘤组织的裂解及各组分的收集
在每只BALB/c小鼠腋下接种2×10 6个4T1乳腺癌细胞，在小鼠所接种肿瘤长到1000mm 3时处死小鼠并摘取肿瘤组织。肺癌细胞收集和处理方法同黑色素瘤癌细胞。肿瘤组织和癌细胞裂解和各组分收集方法同上。将乳腺癌和肺癌的癌细胞裂解物中的水溶性组分按质量比4:1混合，将乳腺癌和肺癌的癌细胞裂解物中的非水溶性组分按质量比4:1混合；然后将水溶性组分混合物和非水溶性组分混合物按质量比1:1混合，即为制备微米疫苗的抗原组分。
(2)微米疫苗的制备
本实施例中制微米疫苗及作为对照的空微米粒采用复乳法制备。微米粒子制备材料PLGA(50:50)分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA(50:50)分子量为24KDa-38KDa。未修饰PLGA和甘露糖修饰的PLGA的质量比为8:2。制备方法如上所述，将抗原组分和佐剂共负载于微米疫苗中。制备方法如前所述。微米疫苗平均粒径为1.50μm左右，平均表面电位为-7mV左右，每1mg PLGA微米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中CpG-ODN 1018为0.025mg，CpG-ODN 1826为0.023mg，Poly(I:C)为0.002mg。空白微米粒粒径为1.45μm左右，空白微米粒制备时负载等量佐剂但不负载抗原组分。对照微米疫苗平均粒径为1.50μm左右，平均表面电位为-7mV左右，每1mg PLGA微米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中CpG-ODN 1018为0.025mg,CpG-ODN 1826为0.024mg,Poly(I:C)为0.001mg。
(3)靶向树突状细胞的微米疫苗用于癌症的预防
选取6-8周的雌性BALB/c小鼠制备乳腺癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天皮下注射200μL的2mg PLGA微米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL PBS。空白微米粒+裂解物对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天 分别皮下注射200μL空白微米粒和与疫苗所负载的等量游离裂解物。在第0天给每只小鼠背部右下方皮下接种4×10 5个4T1乳腺癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图37所示，与对照组相比，疫苗预防组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且，总CpG-ODN与Poly(I:C)质量比25:1作为佐剂的微米疫苗预防效果好于总CpG-ODN与Poly(I:C)质量比49:1作为佐剂的微米疫苗。
实施例10黑色素瘤肿瘤组织和癌细胞全细胞组分负载于纳米疫苗用于黑色素瘤的治疗
本实施例以小鼠黑色素瘤为癌症模型并以Poly(I:C)、CpG-ODN 1018和CpG-ODN 2006为免疫佐剂来说明如何制备负载有黑色素瘤肿瘤组织和癌细胞全细胞组分的纳米疫苗并应用该疫苗治疗黑色素瘤。
本实施例中，首先裂解肝癌和黑色素瘤肿瘤组织的水溶性组分和非水溶性组分并分别按3:1混合。然后，以PLGA为纳米粒子骨架材料采用溶剂挥发法制备纳米疫苗。
(1)肿瘤组织的裂解及各组分的收集
该实施例中肿瘤组织和癌细胞的裂解及裂解物收集同上。非水溶性组分使用5％SDS溶解。将黑色素瘤肿瘤组织和癌细胞的裂解物中的水溶性组分按质量比3:1混合，将黑色素瘤肿瘤组织和癌细胞的非水溶性组分按质量比3:1混合；然后将水溶性组分混合物和非水溶性组分混合物按质量比2:1混合，即为制备纳米疫苗的抗原组分。
(3)纳米疫苗的制备
本实施例中制纳米疫苗及作为对照的空白纳米粒采用复乳法制备。纳米粒子制备材料PLGA(50:50)分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA(50:50)分子量为24KDa-38KDa，未修饰PLGA和甘露糖修饰的PLGA的质量比为9:1。制备方法如上所述，将抗原组分和佐剂共负载于纳米疫苗中。纳米疫苗平均粒径为280nm左右，平均表面电位为-4mV左右，每1mg PLGA纳米疫苗负载95μg蛋白质或多肽组分,每1mg PLGA纳米疫苗使用佐剂0.05mg，其中CpG-ODN 1018为0.025mg,CpG-ODN 2006为0.02mg,Poly(I:C)为0.005mg。对照纳米疫苗1平均粒径为280nm左右，平均表面电位为-5mV左右，每1mg PLGA纳米疫苗负载95μg蛋白质或多肽组分,每1mg PLGA纳米疫苗使用佐剂0.05mg，其中CpG-ODN 1018为0.025mg,CpG-ODN 2006为0.024mg,Poly(I:C)为0.001mg(质量比为25:24:1)。对照纳米疫苗2平均粒径为280nm左右，平均表面电位为-5mV左右，每1mg PLGA纳米疫苗负载95μg蛋白质或多肽组分,每1mg PLGA纳米疫苗使用佐剂0.05mg，其中CpG-ODN 1018为0.02mg,CpG-ODN 2006为0.005mg,Poly(I:C)为0.025mg(质量比为4:1:5)。
(4)纳米疫苗用于黑色素瘤的治疗
选取雌性C57BL/6为模型小鼠制备荷瘤小鼠。各组在在第0天给每只小鼠背部右下方 皮下接种1.5×10 5个B16F10黑色素瘤细胞。疫苗组在肿瘤接种后第4天、第7天、第10天、第15天和第20天分别皮下注射200μL的2mg PLGA纳米疫苗。PBS空白对照组在肿瘤接种后第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。小鼠肿瘤生长监测方法同上。
(4)实验结果
如图38所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且，总CpG-ODN与Poly(I:C)质量比9:1作为佐剂的纳米疫苗预防效果好于总CpG-ODN与Poly(I:C)质量比49:1和1:1作为佐剂的纳米疫苗。
实施例11 8M尿素溶解乳腺癌肿瘤组织组分并负载于纳米粒子治疗乳腺癌
本实施例说明如何采用8M尿素溶解全细胞组分并制备负载有全细胞组分的纳米疫苗以治疗乳腺癌。本实施例中，以4T1小鼠三阴性乳腺癌为癌症模型。首先对乳腺癌肿瘤组织细胞进行灭活和变性处理并以8M尿素裂解肿瘤组织并溶解全细胞组分。然后，以PLGA为纳米粒子骨架材料，以Poly(I:C)、CpG-ODN 1018(B类)和CpG-ODN M362(C类)为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织全细胞组分的纳米疫苗。
(1)肿瘤组织的裂解及各组分的收集
在BALB/c小鼠右腋下皮下接种4×10 5个4T1细胞，在肿瘤长到体积1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后加入胶原酶在37℃作用15分钟，然后研磨通过细胞过滤网过滤并收集过滤所得肿瘤组织细胞。所得肿瘤组织细胞分别采用紫外线和高温加热进行灭活和变性处理，然后采用适量8M尿素裂解乳腺癌肿瘤组织细胞并溶解裂解物，即为制备疫苗的抗原组分来源。
(2)纳米疫苗的制备
本实施例中制纳米疫苗及作为对照的空白纳米粒采用复乳法制备。纳米粒子制备材料PLGA(50:50)分子量为7KDa-17KDa。制备方法如上所述，将抗原组分和佐剂共负载于纳米疫苗中。纳米疫苗平均粒径为250nm左右，平均表面电位为-4mV左右，每1mg PLGA纳米疫苗负载90μg蛋白质或多肽组分,每1mg PLGA纳米疫苗使用佐剂0.048mg，其中CpG-ODN 1018为0.0056mg,CpG-ODN M362为0.04mg,Poly(I:C)为0.0024mg。对照纳米疫苗1平均粒径为250nm左右，平均表面电位为-5mV左右，每1mg PLGA纳米疫苗负载95μg蛋白质或多肽组分，每1mg PLGA纳米疫苗使用佐剂0.048mg，其中CpG-ODN M362为0.047mg,Poly(I:C)为0.001mg(质量比为47:1)。对照纳米疫苗2平均粒径为250nm左右，平均表面电位为-5mV左右，每1mg PLGA纳米疫苗负载90μg蛋白质或多肽组分，不含任何佐剂。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性BALB/c制备4T1荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种4×10 5个4T1细胞。
疫苗治疗组在第4天、第7天、第10天、第15天和第20天皮下注射200μL的2mg PLGA纳米疫苗。
PBS空白对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。
空白纳米粒+游离裂解物对照组在第4天，第7天，第10天，第15天和第20天分别皮下注射等量肿瘤组织裂解物和2mg PLGA空白纳米粒。在实验中，小鼠肿瘤体积监测和计算方法同上。
(4)实验结果
如图39所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且，总CpG-ODN与Poly(I:C)质量比19:1作为佐剂的纳米疫苗预防效果好于总CpG-ODN与Poly(I:C)质量比47:1作为佐剂的纳米疫苗和不含任何佐剂的疫苗。
实施例12黑色素瘤肿瘤组织和肺癌癌细胞全细胞组分负载于纳米疫苗内部和表面用于肺癌的治疗
本实施例说明如何制备负载有黑色素瘤肿瘤组织和肺癌癌细胞的全细胞组分的纳米疫苗，并应用该疫苗治疗肺癌。本实施例中，首先裂解B16F10黑色素瘤肿瘤组织和LLC癌细胞以制备相应的水溶性组分和溶于8M尿素水溶液(含500mM氯化钠)的非水溶性组分。然后将来自肿瘤组织的水溶性组分和来自癌细胞的水溶性组分按质量比1：1混合即为实验中使用的水溶性组分；将来自肿瘤组织的非水溶性组分和来自癌细胞的非水溶性组分按质量比1：1混合即为实验中使用的非水溶性组分；然后，以PLGA为骨架材料，以Poly(I:C)、CpG-ODN 1018(B类)和CpG-ODN 1826(B类)为免疫佐剂制备纳米疫苗。
(1)肿瘤组织及癌细胞的裂解及各组分的收集
B16F10黑色素瘤肿瘤组织和LLC肺癌癌细胞的裂解方法同上，在黑色素瘤肿瘤组织或LLC癌细胞裂解后，将裂解物以8000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分；用8M尿素水溶液(含500mM氯化钠)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8M尿素水溶液(含500mM氯化钠)中可溶。水溶性组分和非水溶性组分分别按照1:1的质量比例混合可得水溶性组分混合物和非水溶性组分混合物，即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗及空白纳米粒采用适当改进的复乳法制备，水溶性组分混合物负载于纳米粒子内部而非水溶性组分混合物负载于纳米疫苗表面，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以Poly(I:C)、CpG-ODN 1018和CpG-ODN 1826分布于纳米粒子内部和表面。在纳米粒子制备过程中采用低温硅化技术和添加带电物质两种修饰方法提高抗原的负载量。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水溶性组分和佐剂，在内部负载抗原和佐剂后，将100mg纳 米粒子在10000g离心20分钟，然后使用7mL PBS重悬纳米粒子并与3mL含有非水溶性组分混合物(60mg/mL)的PBS溶液混合，尔后在10000g离心20分钟，然后采用10mL硅酸盐溶液(含150mM NaCl、80mM原硅酸四甲酯和1.0mM HCl，pH 3.0)重悬,并在室温固定10min，尔后在-80℃固定24h，使用超纯水离心洗涤后使用3mL含鱼精蛋白(5mg/mL)和聚赖氨酸(10mg/mL)的PBS重悬并作用10min,然后10000g离心20min洗涤，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9mLPBS重悬然后加入1mL含免疫佐剂(1mg/mL)的非水溶性组分(蛋白质浓度50mg/mL)室温作用10min，得到内外都负载裂解物组分的经硅化和添加阳离子物质的修饰的纳米粒子系统。该纳米粒子平均粒径为350nm左右，纳米粒子表面电位为-3mV左右；每1mg PLGA纳米粒子约负载250μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的免疫佐剂为0.05mg，其中Poly(I:C)为0.01mg、CpG-ODN 1018(B类)为0.03mg和CpG-ODN 1826(B类)为0.01mg。
对照纳米疫苗制备方法同上，该纳米粒子平均粒径为350nm左右，纳米粒子表面电位为-4mV左右；每1mg PLGA纳米粒子约负载250μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的免疫佐剂为0.05mg，其中Poly(I:C)为0.01mg、CpG-ODN 1585(A类)为0.03mg和CpG-ODN 2216(A类)为0.01mg。
空白纳米粒粒径为320nm左右，空白纳米粒含有等量Poly(I:C)、CpG-ODN 1018和CpG-ODN 1826，但是不含裂解物组分。
(3)纳米疫苗用于肺癌的治疗
选取6-8周的雌性C57BL/6小鼠制备肺癌荷瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1×10 6个LLC肺癌细胞。
疫苗治疗组在第4天、第7天、第10天、第15天和第20天皮下注射200μL的2mg PLGA纳米疫苗。
PBS空白对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。
空白纳米粒+游离裂解物对照组在第4天，第7天，第10天，第15天和第20天分别皮下注射等量肿瘤组织裂解物和2mg PLGA空白纳米粒。在实验中，小鼠肿瘤体积监测和计算方法同上。
(4)实验结果
如图40所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且，使用B类CpG-ODN与Poly(I:C)(质量比4:1)混合作为佐剂的纳米疫苗治疗效果好于使用A类CpG-ODN与Poly(I:C)(质量比4:1)作为佐剂的纳米疫苗。
实施例13结肠癌肿瘤组织全细胞组分负载于纳米疫苗用于结肠癌的治疗
本实施例说明如何制备负载有结肠癌肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗 治疗结肠癌。本实施例中，首先裂解MC38结肠癌肿瘤组织制备相应的水溶性组分和溶于8M尿素的非水溶性组分。将来自肿瘤组织的水溶性组分和非水溶性组分按质量比1：1混合即为实验中使用的裂解物组分；然后，以PLGA为骨架材料，以Poly(I:C)、CpG-ODN 2395(C类)和CpG-ODN SL03(C类)为免疫佐剂，以NH 4HCO 3为增加溶酶体逃逸的物质，制备纳米疫苗。
(1)肿瘤组织及癌细胞的裂解及各组分的收集
选取6-8周的雌性C57BL/6小鼠，在背部接种2×10 6个MC38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适量纯水，使用紫外照射和45℃加热处理，并反复冻融5次，并伴有超声以破坏裂解组织细胞。待组织细胞裂解后，将裂解物以8000g的转速离心5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8M尿素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8M尿素水溶液中可溶。水溶性组分和非水溶性组分按照1:1的质量比例混合可得水溶性组分混合物和非水溶性组分混合物，即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗及空白纳米粒采用适当改进的复乳法制备，水溶性组分混合物负载于纳米粒子内部而非水溶性组分混合物负载于纳米疫苗表面，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以Poly(I:C)、CpG-ODN 2395和CpG-ODN SL03分布于纳米粒子内部和表面，NH 4HCO 3包裹于纳米疫苗内部。制备方法如前所述，在制备过程中首先采用复乳法在纳米粒子内部负载裂解物组分和佐剂，在内部负载抗原和佐剂后，将100mg纳米粒子在10000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。在粒子使用前将其用9mL PBS重悬然后加入1mL含免疫佐剂(1mg/mL)的非水溶性组分(蛋白质浓度50mg/mL)室温作用10min，即可使用。该纳米粒子平均粒径为250nm左右，纳米粒子表面电位为-4mV左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的免疫佐剂为0.04mg，其中Poly(I:C)为0.01mg、CpG-ODN 2395(C类)为0.02mg和CpG-ODN SL03(C类)为0.01mg，负载NH 4HCO 30.05mg。
对照纳米疫苗制备方法同上，该纳米粒子平均粒径为250nm左右，纳米粒子表面电位为-4mV左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的免疫佐剂为0.04mg，其中Poly(I:C)为0.01mg、CpG-ODN 1585(A类)为0.02mg和CpG-ODN 2336(A类)为0.01mg,负载NH 4HCO 30.05mg。
空白纳米粒粒径为240nm左右，空白纳米粒含有等量Poly(I:C)、CpG-ODN 2395(C类)、CpG-ODN SL03(C类)和NH 4HCO 3，但是不含裂解物组分。
(3)纳米疫苗用于结肠癌的治疗
选取6-8周的雌性C57BL/6制备结肠癌荷瘤小鼠。在第0天给每只小鼠背部右下方皮 下接种1×10 6个MC38结肠癌细胞。
疫苗治疗组在第4天、第7天、第10天、第15天和第20天皮下注射200μL的2mg PLGA纳米疫苗。
PBS空白对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。
空白纳米粒+游离裂解物对照组在第4天，第7天，第10天，第15天和第20天分别皮下注射等量肿瘤组织裂解物和2mg PLGA空白纳米粒。在实验中，小鼠肿瘤体积监测和计算方法同上。
(4)实验结果
如图41所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。而且，使用C类CpG-ODN与Poly(I:C)(质量比3:1)混合作为佐剂的纳米疫苗治疗效果好于使用A类CpG-ODN与Poly(I:C)(质量比3:1)作为佐剂的纳米疫苗。
实施例14水溶性细胞组分负载于微米粒子内部和表面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制备只负载有黑色素瘤和肺癌细胞组分中水溶性部分的微米疫苗，并应用该疫苗预防黑色素瘤。本实施例中，首先裂解B16F10黑色素瘤和LLC肺癌细胞以制备水溶性组分和非水溶性组分。然后，以PLGA(24KDa-38KDa)为微米粒子骨架材料，以poly(I:C)和两种CpG-ODN为免疫佐剂制备负载有全细胞的水溶性组分的微米疫苗，并应用该微米疫苗预防黑色素瘤。
(1)癌细胞的裂解及各组分的收集
收集一定量的B16F10细胞或LLC细胞，去除培养基后采用-20℃冷冻，加一定量超纯水后通过加热和紫外照射处理细胞，然后反复冻融3次以上，并伴有超声破坏裂解细胞。待细胞裂解后，将裂解物以8000g的转速离心5min取上清液即为B16F10黑色素瘤或LLC肺癌细胞中可溶于纯水的水溶性组分。上述所得来源于两种癌细胞裂解物的水溶性组分按质量比1:1混合即为制备微米疫苗的抗原来源。
(2)微米疫苗的制备
本实施例中制备微米疫苗及作为对照的空白微米粒采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料为PLGA，所采用的免疫佐剂为poly(I:C)、CpG-ODN 2006(B类)和CpG-ODN2216(A类)，三者质量比为4:0.5:0.5。抗原组分包载于微米疫苗内部和吸附于微米粒子表面，佐剂只包载于微米疫苗内部。制备方法如前所述。在微米粒子表面负载细胞组分和免疫佐剂后所得微米疫苗1粒径为2.10μm，表面电位为-5mV左右，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用佐剂0.05mg，其中Poly(I:C)0.04mg,CpG-ODN 2006 0.005mg,CpG-ODN 2216 0.005mg。对照微米疫苗1粒径为2.10μm，表面电位为-5mV，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用poly(I:C)和CpG-ODN2006作为佐剂，其中poly(I:C)0.04mg,CpG-ODN 2006 0.01mg。对照微米疫苗2 粒径为2.10μm，表面电位为-5mV，每1mg PLGA微米粒子负载150μg蛋白质或多肽组分，使用poly(I:C)、CpG-ODN 2006和CpG-ODN 2216作为佐剂，其中poly(IC)0.025mg,CpG-ODN 2006 0.0125mg，CpG-ODN 2216 0.0125mg。
(3)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备黑色素瘤荷瘤小鼠。微米疫苗组方案如下：在接种黑色素瘤之前第35天、第28天、第21天、第14天分别皮下注射200μL的2mg PLGA微米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。PBS空白对照组方案如下：在接种黑色素瘤之前第35天、第28天、第21天、第14天分别皮下注射200μL PBS；在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。由于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图42所示，与PBS空白对照组，微米疫苗给药组中小鼠肿瘤体积生长速度均明显变慢且小鼠生存期均明显延长。而且，使用poly(I:C)和两种CpG-ODN作为混合佐剂的微米疫苗好于只使用Poly(I:C)和一种CpG-ODN作为混合佐剂的微米疫苗；使用总CpG-ODN:Poly(I:C)＝0.25:1质量比混合作为佐剂的微米疫苗好于使用CpG-ODN:Poly(I:C)＝1:1质量比混合作为佐剂的微米疫苗。
实施例15佐剂和黑色素瘤癌细胞负载于纳米粒子内部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制备共负载免疫佐剂和黑色素瘤癌细胞全细胞组分的纳米疫苗，并应用该疫苗治疗黑色素瘤。本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。首先裂解B16F10黑色素瘤癌细胞并制备水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以poly(I:C)、CpG-ODN 1018、CpG-ODN 2336和精氨酸为免疫佐剂采用溶剂挥发法制备负载有癌细胞的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗黑色素瘤。
(1)细胞的裂解及各组分的收集
将培养的B16F10癌细胞系去除培养基后在400g离心5分钟，使用PBS重悬并洗涤两遍，然后使用超纯水重悬癌细胞，并反复冻融五次，冻融过程中使用超声辅助裂解癌细胞，然后在10000g离心5分钟，收集上清液即为水溶性组分。将沉淀部分使用8M尿素溶解，即为溶解的原非水溶性组分。将来自B16F10癌细胞的水溶性组分和溶解于8M尿素中的原非水溶性组分按照质量比1:1的比例混合即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa，佐剂和抗原组分包载于纳米疫苗中，所采用的免疫佐剂为poly(I:C)、CpG-ODN 1018、CpG-ODN 2336和精氨酸。制备方法如前所述。负载全细胞组 分的纳米疫苗平均粒径为280nm左右；纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的佐剂为0.05mg，其中poly(I:C)为0.02mg,CpG-ODN 1018为0.01mg,CpG-ODN 2336为0.02mg，使用精氨酸为0.5mg。对照纳米疫苗1的制备方法相同，粒径为280nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mgPLGA纳米粒使用佐剂为0.05mg，poly(I:C)为0.02mg,CpG-ODN 1018为0.015mg,CpG-ODN 2336为0.015mg。对照纳米疫苗2的制备方法相同，粒径为280nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mgPLGA纳米粒使用0.5mg精氨酸作为免疫佐剂。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。纳米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞；在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射100μL负载水溶性成分的2mg PLGA纳米疫苗。PBS对照组方案如下：在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞；在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。小鼠肿瘤监测方案同上。
(4)实验结果
如图43所示，PBS对照组小鼠的肿瘤都长大且生产速度很快。纳米疫苗组的疗效都好于PBS对照组，而且使用两种以上CpG+Poly(I:C)+精氨酸作为混合佐剂的纳米疫苗好于使用两种以上CpG+Poly(I:C)作为混合佐剂的纳米疫苗，也好于只使用精氨酸作为佐剂的纳米疫苗。本发明所述的负载特定比例的多种CpG和Poly(I:C)及抗原组分的纳米疫苗对黑色素瘤具有良好的治疗效果。
实施例16佐剂和乳腺癌癌细胞负载于纳米粒子内部用于乳腺癌的治疗
本实施例以小鼠三阴性乳腺癌为癌症模型来说明如何制备共负载免疫佐剂、增加溶酶体逃逸的物质和三阴性乳腺癌癌细胞全细胞组分的纳米疫苗，并应用该疫苗治疗三阴性乳腺癌。本实施例中，首先裂解4T1乳腺癌癌细胞并制备水溶性组分和非水溶性组分。然后，以PLGA为纳米粒骨架材料，以Poly(I:C)、CpG-ODN 1018、CpG-ODN 2336为免疫佐剂，以赖氨酸为增加溶酶体逃逸物质，采用溶剂挥发法制备负载有癌细胞的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗治疗乳腺癌。
(1)细胞的裂解及各组分的收集
将培养的4T1癌细胞系去除培养基后在400g离心5分钟，使用PBS重悬并洗涤两遍，然后使用超纯水重悬癌细胞，并反复冻融五次，冻融过程中使用超声辅助裂解癌细胞，加入核酸酶作用10分钟降解所有的核酸，然后在95℃加热5分钟灭活核酸酶，然后在10000g离心5分钟，收集上清液即为水溶性组分。将沉淀部分使用8M尿素溶解，即为溶解的原非水溶性组分。将来自癌细胞的水溶性组分和溶解于8M尿素中的原非水溶性组分按照质 量比2:1的比例混合即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为7KDa-17KDa，佐剂和抗原组分包载于纳米疫苗中，所采用的免疫佐剂为poly(I:C)、CpG-ODN 1018、CpG-ODN 2336和赖氨酸。制备方法如前所述。负载全细胞组分的纳米疫苗平均粒径为280nm左右；纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分，每1mg PLGA纳米粒使用的佐剂为0.05mg，其中poly(I:C)为0.02mg,CpG-ODN 1018为0.015mg,CpG-ODN 2216为0.015mg，使用赖氨酸0.005mg。对照纳米疫苗1的制备方法相同，粒径为280nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mgPLGA纳米粒使用的佐剂为0.05mg，其中poly(I:C)为0.02mg,CpG-ODN 1018为0.015mg,CpG-ODN 2216为0.015mg,使用甘氨酸0.005mg。对照纳米疫苗2的制备方法相同，粒径为280nm，疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分；每1mgPLGA纳米粒使用佐剂为0.05mg，其中poly(I:C)为0.02mg,CpG-ODN 1585为0.015mg,CpG-ODN 2216为0.015mg,使用赖氨酸0.005mg。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性BALB/c为模型小鼠制备乳腺癌荷瘤小鼠。纳米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种4×10 5个4T1细胞；在接种乳腺癌后第4天、第7天、第10天、第15天和第20天分别皮下注射100μL负载水溶性成分的2mg PLGA纳米疫苗。PBS对照组方案如下：在第0天给每只小鼠背部右下方皮下接种4×10 5个4T1细胞；在接种乳腺癌后第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。小鼠肿瘤监测方案同上。
(4)实验结果
如图44所示，PBS对照组小鼠的肿瘤都长大且生产速度很快。纳米疫苗组的疗效都好于PBS对照组，而且使用两种以上CpG-ODN(A类+B类)+Poly(I:C)+赖氨酸作为混合佐剂的纳米疫苗好于使用两种以上CpG-ODN(A类+B类)+Poly(I:C)作为混合佐剂的纳米疫苗，也好于使用两种以上CpG-ODN(A类+A类)+Poly(I:C)+赖氨酸作为佐剂的纳米疫苗。本发明所述的负载特定比例的多种CpG和Poly(I:C)及抗原组分的纳米疫苗对乳腺癌具有良好的治疗效果。
实施例17全细胞组分负载于甘露聚糖修饰的微米粒子内部用于乳腺癌预防
本实施例以小鼠乳腺癌为癌症模型来说明如何制备负载有乳腺癌肿瘤组织和癌细胞的全细胞组分的微米疫苗，并应用该疫苗预防乳腺癌。在实际应用时粒子大小，给药时间、给药次数、给药方案可根据情况调整。本实施例中，首先取得小鼠乳腺癌的肿瘤组织和癌细胞并将其裂解以制备水溶性组分和溶于8M尿素中的原非水溶性组分。然后，以PLGA和甘露聚糖修饰的PLGA为微米粒骨架材料，以Poly ICLC、CpG-ODN 1018(B类)和 CpG-ODN 2395(C类)为免疫佐剂，以TAT多肽(YGRKKRRQRRR)增加免疫逃逸的物质，采用溶剂挥发法制备微米疫苗。TAT多肽含有带正电荷的赖氨酸和精氨酸。该微米疫苗具有靶向树突状细胞的能力。
(1)肿瘤组织的裂解及各组分的收集
在每只BALB/c小鼠腋下接种4×10 5个4T1乳腺癌细胞，在小鼠所接种肿瘤长到1000mm 3时处死小鼠并摘取肿瘤组织。4T1癌细胞和癌肿瘤组织的裂解和各组分收集方法同上。将乳腺癌肿瘤组织和4T1癌细胞裂解物中的水溶性组分按质量比2:1混合，将乳腺癌肿瘤组织和4T1癌细胞裂解物中的非水溶性组分按质量比2:1混合；然后将水溶性组分混合物和非水溶性组分混合物按质量比1:2混合，即为制备微米疫苗的抗原组分。
(2)微米疫苗的制备
本实施例中制微米疫苗及作为对照的空微米粒采用复乳法制备。微米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的甘露聚糖修饰的PLGA分子量为24KDa-38KDa；未修饰的PLGA和甘露聚糖修饰的PLGA的质量比为8:2。制备方法如上所述，将裂解物组分和佐剂共负载于微米疫苗内。微米疫苗平均粒径为1.50μm左右，平均表面电位为-7mV左右，每1mg PLGA微米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中Poly ICLC 0.01mg、CpG-ODN 1018(B类)0.02mg、CpG-ODN 2395(C类)0.02mg，TAT多肽0.1mg。空白微米粒制备所用聚合物材料和制备方法同上，粒径为1.45μm左右，空白微米粒制备时负载等量佐剂和TAT多肽但不负载抗原组分。对照微米疫苗制备所用聚合物材料和制备方法同时，平均粒径为1.50μm左右，平均表面电位为-7mV左右，每1mg PLGA微米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中Poly ICLC为0.01mg、CpG-ODN 1585(A类)为0.01mg、CpG-ODN 2336(A类)为0.01mg，TAT多肽为0.1mg。
(3)靶向树突状细胞的微米疫苗用于癌症的预防
选取6-8周的雌性BALB/c小鼠制备乳腺癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天皮下注射200μL的2mg PLGA微米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL PBS。空白微米粒+裂解物对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL空白微米粒和与疫苗所负载的等量游离裂解物。在第0天给每只小鼠背部右下方皮下接种4×10 5个4T1乳腺癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图45所示，与对照组和空白微米粒+游离裂解液组相比，疫苗预防组肿瘤生长速度和小鼠生存期都有显著性差异。而且，本实施例所制备的微米疫苗好于对照微米疫苗组，这说明使用C类CpG-ODN与B类CpG-ODN混合作为佐剂效果优于两种A类CpG-ODN混合。
实施例18肝癌肿瘤组织裂解组分负载于微米粒子用于预防肝癌
本实施例制备负载有肝癌和肺癌肿瘤组织裂解物组分的微米疫苗，并应用该疫苗预防肝癌。本实施例中，将小鼠肝癌肿瘤组织裂解组分负载于微米疫苗。首先取得小鼠肝癌和肺癌肿瘤组织，使用8M尿素裂解肿瘤组织并溶解肿瘤组织裂解物组分。然后，以PLA(30KDa)为微米粒骨架材料，以Poly ICLC、CpG-ODN 2007(B类)和CpG-ODN2216(A类)为免疫佐剂，以cR8(cyclo(CRRRRRRRRC))多肽为增加免疫逃逸物质，制备微米疫苗,并应用该微米疫苗预防肝癌。cR8为含有精氨酸的环状多肽。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种2×10 6个Hepa 1-6细胞或者2×10 6个LLC肺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切碎过细胞筛网，然后使用8M尿素裂解肿瘤组织后溶解；将肝癌肿瘤组织裂解物和肺癌肿瘤组织裂解物按质量比1:1混合即为制备疫苗所使用的抗原组分。
(2)微米疫苗的制备
本实施例中微米疫苗采用溶剂挥发法制备，所采用制备材料为PLA，所采用的免疫佐剂为Poly ICLC、CpG-ODN 2007、CpG-ODN 2216，四者质量比为1:1:1；所使用的增加溶酶体逃逸的物质为cR8多肽，cR8多肽五分之一通过化学键修饰PLA位于粒子表面，五分之四包载于粒子内部。抗原组分包载于微米疫苗内部和吸附于微米粒子表面，佐剂和增加溶酶体逃逸的物质只包载于微米疫苗内部。制备时先采用溶解挥发法将抗原组分和佐剂包载于微米粒子内部，然后在8000g离心15min,收集沉淀并将100mg PLA微米粒子使用4％海藻糖溶液重悬，然后冷冻干燥48小时后冷藏备用。在注射使用前，将10mg PLA微米粒子使用0.9mL PBS或生理盐水重悬，然后与0.1mL含有8M尿素中的细胞裂解液(60mg/mL)和免疫佐剂(3mg/mL,四者质量比1:1:1)的样品在室温混合作用10min,即可注射使用。所得微米疫苗粒径为3.10μm，每1mg PLA微米粒子负载150μg蛋白质或多肽组分，使用佐剂0.045mg，其中Poly ICLC 0.015mg，CpG-ODN2007 0.015mg，CpG-ODN 2216 0.015mg；使用cR8多肽0.02mg，其中0.004mg通过化学修饰与PLA相连位于粒子表面，0.016mg包载于粒子内部。对照微米疫苗1粒径为3.10μm，每1mg PLA微米粒子负载150μg蛋白质或多肽组分，使用Poly ICLC、CpG-ODN 1585和CpG-ODN 2216各0.015mg；使用cR8多肽0.02mg，其中0.004mg通过化学修饰与PLA相连位于粒子表面，0.016mg包载于粒子内部。对照微米疫苗2只负载免疫佐剂的空白微米粒粒径为3.00μm，每1mg PLA微米粒子负载150μg蛋白质或多肽组分，使用Poly ICLC 0.015mg,CpG-ODN 2007 0.03mg；使用cR8多肽0.02mg，其中0.004mg通过化学修饰与PLA相连位于粒子表面，0.016mg包载于粒子内部。
(3)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备Hepa 1-6肝癌荷瘤小鼠。在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL的2mg PLA微米疫苗。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。PBS空白对照组方案 如下：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL PBS。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图46所示，PBS对照组小鼠的肝癌肿瘤生长均较快，微米疫苗给药组都可显著抑制小鼠肿瘤生长和延长小鼠生存期。而且，本实施例所使用的疫苗效果好于对照疫苗1和对照疫苗2。由此可见，同时使用B类CpG-ODN和A类CpG-ODN与Poly ICLC作为混合佐剂好于只使用B类CpG-ODN与Poly ICLC作为混合佐剂和同时使用两种A类CpG-ODN与Poly ICLC作为混合佐剂。
实施例19肿瘤组织裂解组分负载于纳米粒子内部用于胰腺癌的治疗
本实施例以小鼠胰腺癌为癌症模型来说明如何制备负载有胰腺癌肿瘤组织裂解物组分的纳米疫苗，并应用该疫苗治疗胰腺癌。首先取得小鼠胰腺癌肿瘤组织并将其裂解以制备水溶性组分和溶于6M盐酸胍中的原非水溶性组分。以PLGA(分子量7KDa-17KDa)为粒子骨架材料，以Poly(I:C)、CpG-ODN M362、CpG-ODN 2336为免疫佐剂,以HER2多肽(YDLKEPEH)为增加溶酶体逃逸物质，制备纳米疫苗。HER2多肽内含有组氨酸和精氨酸。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种1×10 6个Pan02胰腺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。肿瘤组织的裂解方法同实施例18，收集水溶性组分后，使用6M盐酸胍溶解非水溶性组分。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗采用溶剂挥发法，PLGA分子量为24KDa-38KDa，以Poly(I:C)、CpG-ODN M362、CpG-ODN 2336按质量比1:0.8:0.8作为混合佐剂使用，以HER2多肽为增加溶酶体逃逸物质。在制备疫苗时，水溶性组分混合物和非水溶性组分混合物分别制备成纳米疫苗再一起使用，抗原组分和佐剂只包载于纳米疫苗内。制备方法同上。纳米疫苗粒径为270nm左右，表面电位为-7mV左右，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.026mg，其中Poly(I:C)为0.01mg，CpG-ODN M362为0.008mg：CpG-ODN 2336为0.008mg；使用HER2多肽0.026mg。对照纳米疫苗1粒径为280nm左右，表面电位为-8mV左右，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.026mg,其中Poly(I:C)为0.01mg，CpG-ODN M362为0.08mg：CpG-ODN 2336为0.08mg。对照纳米疫苗2粒径为280nm左右，表面电位为-8mV左右，每1mg PLGA纳米粒子约负载80μg蛋白质或多肽组分,每1mg PLGA纳米粒使用佐剂共0.026mg,Poly(I:C)为0.01mg,其中CpG1585为0.08mg，CpG-ODN 2216为0.08mg，使用HER2多肽0.026mg。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6制备胰腺癌瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1×10 6个个Pan02细胞。疫苗组在第4天、第7天、第10天、第15天和第20天分别皮下注射100μL负载水溶性成分的1mg PLGA纳米疫苗和100μL负载原非水溶性成分的1mg PLGA纳米疫苗。PBS空白对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。在实验中，小鼠肿瘤监测和体积计算方法同上。
(4)实验结果
如图47所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后部分消失。本实施例所述疫苗好于对照纳米疫苗1和对照纳米疫苗2。综上所述，本发明所述的负载特定比例的多种CpG和Poly(I:C)混合佐剂及抗原组分的纳米疫苗对癌症具有良好的治疗效果，而且加入含带正电荷氨基酸的多肽可以提高疫苗效果，而且，一种C类CpG-ODN和一种A类CpG-ODN混用效果好于使用两种A类CpG-ODN。
实施例20纳米疫苗通过细胞免疫中的T细胞免疫发挥癌症治疗功效
本实施例以小鼠黑色素瘤为癌症模型来说明纳米疫苗主要通过细胞免疫中的T细胞免疫来发挥功效。本实施例中，首先裂解B16F10黑色素瘤癌细胞并制备水溶性组分和非水溶性组分。然后，以PLGA为纳米粒骨架材料，以poly(I:C)、CpG-ODN 1018(B类)、CpG-ODN 2336(A类)为免疫佐剂，以精氨酸和聚组氨酸为增加溶酶体逃逸的物质，采用溶剂挥发法制备负载有癌细胞的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗黑色素瘤。
(1)细胞的裂解及各组分的收集
将培养的B16F10癌细胞系去除培养基后在400g离心5分钟，使用PBS重悬并洗涤两遍，然后使用超纯水重悬癌细胞，并反复冻融五次，冻融过程中使用超声辅助裂解癌细胞，然后在10000g离心5分钟，收集上清液即为水溶性组分。将沉淀部分使用8M尿素溶解，即为溶解的原非水溶性组分。将来自B16F10癌细胞的水溶性组分和溶解于8M尿素中的原非水溶性组分按照质量比1:1的比例混合即为制备疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗采用溶剂挥发法制备，所采用PLGA分子量为7KDa-17KDa，佐剂、裂解组分及精氨酸和聚组氨酸包载于纳米疫苗内，所采用的免疫佐剂为poly(I:C)、CpG-ODN 1018、CpG-ODN 2336，增加免疫逃逸的物质为精氨酸和聚组氨酸。制备方法如前所述。负载全细胞组分的纳米疫苗平均粒径为280nm左右；纳米疫苗表面电位为-8mV左右；每1mg PLGA纳米粒子约负载90μg蛋白质或多肽组分，每1mgPLGA纳米粒使用的佐剂为0.05mg，其中poly(I:C)为0.02mg,CpG-ODN 1018为0.015mg,CpG-ODN 2336为0.015mg，负载精氨酸为0.005mg，负载聚组氨酸为0.005mg。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。纳米疫苗组给药方案 如下：在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞；在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射100μL的2mg PLGA纳米疫苗；CD4 +T细胞拮抗组除了给予疫苗外，从接种癌细胞第0天的前两天开始每天腹腔注射10mg/kg的αCD4抗体以清除小鼠体内的CD4 +T细胞，连续给与抗体至第50天；CD8 +T细胞拮抗组除了给予疫苗外，从接种癌细胞第0天的前两天开始每天腹腔注射10mg/kg的αCD8抗体以清除小鼠体内的CD8 +T细胞，连续给与抗体至第50天。PBS对照组方案如下：在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞；在接种黑色素瘤后第4天、第7天、第10天、第15天和第20天分别皮下注射200μL PBS。小鼠肿瘤监测方案同上。
(4)实验结果
如图48所示，PBS对照组小鼠的肿瘤都长大且生产速度很快，疫苗组的小鼠肿瘤生长速度和生存期都明显好于PBS对照组。使用αCD4抗体耗竭掉小鼠体内CD4 +T细胞后，癌症疫苗对癌症仍有很强的治疗作用；使用αCD8抗体耗竭掉小鼠体内CD8 +T细胞后，癌症疫苗丧失了大部分功效对癌症仅有较弱的治疗作用。这说明，本发明所述疫苗主要通过细胞免疫中的T细胞免疫发挥功效。
实施例21微米疫苗通过细胞免疫中的T细胞免疫发挥预防癌症的功效
本实施例以小鼠乳腺癌为癌症模型来说明微米疫苗主要通过T细胞发挥作用。本实施例中，首先取得小鼠乳腺癌肿瘤组织，并将其以8M尿素裂解后溶于8M尿素中。然后，以PLGA和甘露糖修饰的PLGA为微米粒骨架材料，以Poly(I:C)、CpG-ODN 1018(B类)和CpG-ODN 2216(A类)为免疫佐剂采用溶剂挥发法制备微米疫苗。
(1)肿瘤组织的裂解及各组分的收集
在每只BALB/c小鼠腋下接种4×10 5个4T1乳腺癌细胞，在小鼠所接种肿瘤长到1000mm 3时处死小鼠并摘取肿瘤组织。肿瘤组织切成小块后通过细胞筛网过滤制备单细胞悬液，使用8M尿素裂解细胞后溶解裂解后的组分，即为制备微米疫苗的抗原组分。
(2)微米疫苗的制备
本实施例中微米疫苗及作为对照的空微米粒采用复乳法制备。微米粒子制备材料PLGA分子量为38KDa-54KDa，所采用的甘露糖修饰的PLGA分子量也为38KDa-54KDa。未修饰PLGA和甘露糖修饰的PLGA的质量比为8:2。制备方法如上所述，将裂解物组分和佐剂共负载于微米疫苗中。制备方法如前所述。微米疫苗平均粒径为2.50μm左右，平均表面电位为-9mV左右，每1mg PLGA微米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中CpG-ODN 1018为0.02mg，CpG-ODN 2216为0.01mg，Poly(I:C)为0.002mg。
(3)靶向树突状细胞的微米疫苗用于癌症的预防
选取6-8周的雌性BALB/c小鼠制备乳腺癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天皮下注射200μL的2mg PLGA微米疫苗；在第0天 给每只小鼠背部右下方皮下接种4×10 5个4T1乳腺癌细胞；CD4 +T细胞和CD8 +T细胞同时拮抗组除了给予疫苗外，从接种癌细胞第0天的前两天开始每天腹腔注射10mg/kg的αCD4抗体和10mg/kg的αCD8抗体以清除小鼠体内的CD4 +T细胞和CD8 +T细胞，连续给与抗体至第50天；CD8 +T细胞拮抗组除了给予疫苗外，从接种癌细胞第0天的前两天开始每天腹腔注射10mg/kg的αCD8抗体以清除小鼠体内的CD8 +T细胞，连续给与抗体至第50天。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL PBS；在第0天给每只小鼠背部右下方皮下接种4×10 5个4T1乳腺癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图49所示，与对照组相比，疫苗预防组肿瘤生长速度和小鼠生存期都有显著性差异。使用αCD4抗体耗竭掉小鼠体内CD4 +T细胞后，癌症疫苗对癌症仍有很强的预防作用；使用αCD8抗体耗竭掉小鼠体内CD8 +T细胞后，癌症疫苗丧失了大部分功效对癌症仅有较弱的预防作用。这说明，本发明所述疫苗主要通过细胞免疫中的T细胞免疫发挥功效。
实施例22全细胞组分负载于甘露糖修饰的微米粒子用于乳腺癌治疗
本实施例以小鼠乳腺癌为癌症模型来说明如何制备负载有乳腺癌肿瘤组织的全细胞组分的微米疫苗，并应用该疫苗治疗乳腺癌。在实际应用时粒子大小，给药时间、给药次数、给药方案可根据情况调整。本实施例中，以PLGA和甘露糖修饰的PLGA为微米粒骨架材料，以Poly ICLC、CpG-ODN 1018(B类)和CpG-ODN 2395(C类)为免疫佐剂，以精氨酸和赖氨酸混合物为增加免疫逃逸物质，制备微米疫苗。该微米疫苗具有靶向树突状细胞的能力。
(1)肿瘤组织的裂解及各组分的收集
在每只BALB/c小鼠腋下接种4×10 5个4T1乳腺癌细胞，在小鼠所接种肿瘤长到1000mm 3时处死小鼠并摘取肿瘤组织。4T1癌肿瘤组织的裂解和各组分收集方法同上。使用8M尿素溶解非水溶性组分。将乳腺癌肿瘤组织的水溶性组分和非水溶性组分按质量比1:1混合，即为制备微米疫苗的抗原组分。
(2)微米疫苗的制备
本实施例中制微米疫苗及作为对照的空微米粒采用复乳法制备。微米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA分子量为24KDa-38KDa；未修饰的PLGA和甘露糖修饰的PLGA的质量比为7:3。制备方法如上所述，将裂解物组分和佐剂共负载于微米疫苗内。微米疫苗平均粒径为1.50μm左右，平均表面电位为-7mV左右，每1mg PLGA微米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中Poly ICLC 0.01mg、CpG-ODN 1018(B类)0.02mg、CpG-ODN 2395(C类)0.02mg，使用精氨酸0.05mg，赖氨酸0.05mg。对照微米疫苗1粒径为1.50μm左右，平均表面电位为-7mV左右，负载等量佐剂和细胞裂解物组分，负载0.1mg精氨酸。对照微米疫苗2平均粒径为1.50μm左右，平均表面电位为-7mV左右，每1mg PLGA微 米粒子负载85μg蛋白质或多肽组分,每1mg PLGA微米疫苗使用佐剂0.05mg，其中Poly ICLC为0.01mg、CpG-ODN 1018(B类)0.02mg、CpG-ODN 2395(C类)0.02mg，负载赖氨酸0.1mg。
(3)靶向树突状细胞的微米疫苗用于癌症的治疗
选取6-8周的雌性BALB/c小鼠制备乳腺癌荷瘤小鼠。第0天给每只小鼠背部右下方皮下接种4×10 5个4T1乳腺癌细胞。疫苗组在肿瘤接种后第4天、第7天、第10天、第14天、第19天和第24天皮下注射200μL的2mg PLGA微米疫苗。PBS空白对照组在肿瘤接种后第4天、第7天、第10天、第14天、第19天和第24天分别皮下注射200μL PBS。在在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图50所示，与对照组相比，疫苗治疗组肿瘤生长速度和小鼠生存期都有显著性差异。而且，本实施例所制备的微米疫苗好于对照微米疫苗组，这说明使用混合氨基酸效果好于使用单个氨基酸。
实施例23肝癌肿瘤组织裂解组分负载于微米粒子用于治疗肝癌
本实施例制备负载有肝癌肿瘤组织裂解物组分的微米疫苗，并应用该疫苗预防肝癌。本实施例中，以PLGA(38KDa-54KDa)为微米粒骨架材料，以Poly ICLC、CpG-ODN 2007(B类)和CpG-ODN2216(A类)为免疫佐剂，以组氨酸为促进溶酶体逃逸的物质，制备微米疫苗,并应用该微米疫苗治疗肝癌。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种2×10 6个Hepa 1-6肝癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切碎过细胞筛网，然后使用6M盐酸胍裂解肿瘤组织后溶解裂解组分即为制备疫苗所使用的的抗原组分。
(2)微米疫苗的制备
本实施例中微米疫苗采用溶剂挥发法制备，所采用制备材料为PLGA，所采用的免疫佐剂为Poly ICLC、CpG-ODN 2007、CpG-ODN 2216，四者质量比为1:1:1；所使用的增加溶酶体逃逸的物质为组氨酸。抗原组分、佐剂和增加溶酶体逃逸的物质包载于微米疫苗内部。制备时先采用溶解挥发法将抗原组分和佐剂包载于微米粒子内部，然后在8000g离心15min,收集沉淀并将100mg PLA微米粒子使用4％海藻糖溶液重悬，然后冷冻干燥48小时后冷藏备用。所得微米疫苗粒径为2.60μm，每1mg PLGA微米粒子负载100μg蛋白质或多肽组分，使用佐剂0.045mg，其中Poly ICLC 0.015mg，CpG-ODN 2007 0.015mg，CpG-ODN 2216 0.015mg；使用组氨酸0.02mg。对照微米疫苗1粒径为2.60μm，每1mg PLGA微米粒子负载100μg蛋白质或多肽组分，使用Poly ICLC、CpG-ODN 2007和CpG-ODN 2216各0.015mg；使用谷氨酸0.02mg。对照微米疫苗2粒径为2.60μm，每1mg PLGA微米粒子负载100μg蛋白质或多肽组分，使用Poly ICLC 0.015mg,CpG-ODN 2007 0.015mg，CpG-ODN 2216 0.015mg；使用含有组氨酸的8肽(LHQAVPGL)0.02mg。
(3)微米疫苗用于癌症的治疗
选取6-8周的雌性C57BL/6制备Hepa 1-6肝癌荷瘤小鼠。在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。在接种肝癌细胞后第4天、第7天、第10天、第15天、第20和第25天天分别皮下注射200μL的2mg PLA微米疫苗。在实验中，小鼠肿瘤生长监测方法同上。
(4)实验结果
如图51所示，PBS对照组小鼠的肝癌肿瘤生长均较快，微米疫苗给药组都可显著抑制小鼠肿瘤生长和延长小鼠生存期。而且，本实施例所使用的疫苗效果好于对照疫苗1和对照疫苗2。由此可见，使用带正电的氨基酸效果好于带负电的氨基酸，而且，纯组氨酸效果好于含部分组氨酸酸的多肽。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (14)

  1. 一种免疫佐剂组合物，其特征在于，所述免疫佐剂组合物至少包括以下组分中的(1)和(2)的组合：
    (1)Poly(I:C)或Poly(ICLC)，
    (2)CpG-ODN，其中，所述CpG-ODN为A类CpG-ODN、B类CpG-ODN和C类CpG-ODN中的至少两种，且至少其中一种为B类CpG-ODN或C类CpG-ODN；
    (3)氨基酸、多肽、脂类、糖类、蛋白质或无机盐。
  2. 根据权利要求1所述的免疫佐剂组合物，其特征在于：所述A类CpG-ODN选自CpG-ODN 2216、CpG-ODN 1585或CpG-ODN 2336。
  3. 根据权利要求1所述的免疫佐剂组合物，其特征在于：所述B类CpG-ODN选自CpG-ODN 1018、CpG-ODN 2006、CpG-ODN 1826、CpG-ODN 1668、CpG-ODN 2007、CpG-ODN BW006或CpG-ODN SL01。
  4. 根据权利要求1所述的免疫佐剂组合物，其特征在于：所述C类CpG-ODN选自CpG-ODN 2395、CpG-ODN SL03或CpG-ODN M362。
  5. 根据权利要求1所述的免疫佐剂组合物，其特征在于：所述氨基酸为带正电的氨基酸。
  6. 根据权利要求1所述的免疫佐剂组合物，其特征在于：所述氨基酸为至少包括两种带正电的氨基酸的组合。
  7. 根据权利要求1所述的免疫佐剂组合物，其特征在于：所述无机盐为可释放H +或酸性物质以形成质子海绵效应的无机盐。
  8. 权利要求1-7任一项所述的免疫佐剂组合物在制备细胞免疫激活剂中的应用。
  9. 一种细胞免疫激活剂，其特征在于：所述细胞免疫激活剂包括权利要求1-7任一项所述的免疫佐剂组合物。
  10. 一种负载权利要求1-7任一项所述的免疫佐剂组合物的癌症疫苗，其特征在于：所述癌症疫苗包括纳米粒子或微米粒子，以及负载于所述纳米粒子或微米粒子上的抗原组分和免疫佐剂组合物。
  11. 根据权利要求10所述的癌症疫苗，其特征在于：所述抗原组分为来源于癌细胞和/或肿瘤组织的全细胞组分抗原。
  12. 根据权利要求11所述的癌症疫苗，其特征在于，所述全细胞组分抗原的制备方法包括以下步骤：
    将癌细胞或肿瘤组织用水或不含溶解剂的溶液裂解，收集可溶部分，为水溶性组分，将不可溶部分用溶解剂溶解后转为可溶的部分为非水溶性组分，所述水溶性组分和非水溶性组分为全细胞组分抗原；
    或将癌细胞或肿瘤组织用溶解剂裂解，再将裂解物溶解，得到所述全细胞组分抗原；
    其中，溶解剂选自尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、0.1-2000mg/mL的无机盐、Triton、吐温、二甲基亚砜、乙腈、乙醇、甲醇、N,N-二甲基甲酰胺、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷、胆碱、Brij TM-35、Octaethylene glycol monododecyl ether、CHAPS、Digitonin、lauryldimethylamine oxide和
    Figure PCTCN2022092201-appb-100001
    CA-630非离子型表面活性剂中的至少一种。
  13. 根据权利要求10所述的癌症疫苗，其特征在于：所述癌症疫苗上连接具有主动靶向功能的靶头。
  14. 权利要求1-7任一项所述的免疫佐剂组合物或权利要求10-13任一项所述的癌症疫苗在制备癌症治疗药物或癌症预防药物中的应用。
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