WO2023040127A1 - Use of cancer vaccine system based on whole cell components in preparation of drugs for cross-prevention or treatment of heterogeneous cancers - Google Patents

Use of cancer vaccine system based on whole cell components in preparation of drugs for cross-prevention or treatment of heterogeneous cancers Download PDF

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Publication number
WO2023040127A1
WO2023040127A1 PCT/CN2021/143434 CN2021143434W WO2023040127A1 WO 2023040127 A1 WO2023040127 A1 WO 2023040127A1 CN 2021143434 W CN2021143434 W CN 2021143434W WO 2023040127 A1 WO2023040127 A1 WO 2023040127A1
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water
components
cancer
vaccine
whole cell
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PCT/CN2021/143434
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French (fr)
Chinese (zh)
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刘密
刁璐
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苏州尔生生物医药有限公司
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Publication of WO2023040127A1 publication Critical patent/WO2023040127A1/en

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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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the field of immunotherapy, in particular to a broad-spectrum nano or micro cancer vaccine based on cancer cells or tumor tissues, in particular to a whole cell based on cancer cells and/or tumor tissues of one or more cancers Components of broad-spectrum nano- or micro-cancer vaccines and their use in the cross-prophylaxis of many other different types of cancer.
  • Immunity is a physiological function of the human body.
  • the human body relies on this function to identify "self” and “non-self” components, thereby destroying and repelling antigenic substances (such as viruses and bacteria) entering the human body, or damaged cells produced by the human body itself and tumor cells to maintain human health.
  • Immunotechnology has developed extremely rapidly in recent years, especially in the field of cancer immunotherapy. With the continuous improvement of the understanding of cancer, people have found that the human immune system and various immune cells play a key role in the process of inhibiting the occurrence and development of cancer. By regulating the strength and balance of the body's immune system, it is expected to affect and control the occurrence, development and treatment of cancer.
  • cancer vaccines have shown great potential in the prevention and treatment of cancer.
  • the basis for developing cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormally mutated cancer cells, and cancer cells or cancer tumor tissues themselves are the best source of cancer antigens.
  • scientists have used new technologies to analyze and identify cancer-specific or cancer-related antigenic polypeptides from tumor cells of cancer patients, and then artificially synthesize them in vitro to prepare cancer vaccines for cancer treatment.
  • the technology has shown some efficacy in clinical trials of cancer patients, but such methods are time-consuming, laborious and costly.
  • the methods used only extract and analyze the differences between cancer cells and normal cells from the water-soluble components of cancer cells, and then look for the different polypeptides. Therefore, such methods and technologies can only find a limited number of antigens with good water solubility. Peptides, which greatly limit the application of this type of method.
  • many antigenic proteins or polypeptides with strong immunogenicity in the real environment of the human body are insoluble in pure water. Water-insoluble proteins and peptides in water are very important and critical. Therefore, it is a promising approach to use cancer cells or whole cell components of cancer tissues as a source of vaccines for the prevention and treatment of cancer.
  • the prior art discloses a targeted delivery system loaded with whole cell components, which is a nano-sized or micron-sized particle with a target head on the surface, and the particles are loaded with whole cell components of cancer cells or tumor tissues ;
  • the whole cell component is the water-soluble component and the water-insoluble component of the whole cell in the cell or tissue, and the water-insoluble component is dissolved by a solubilizing agent;
  • the target head is combined with molecules on the surface of a specific cell or tissue to help The particles enter cells or tissues; however, the disclosed delivery system is used for the prevention and treatment of the same cancer.
  • the object of the present invention is to address the problems existing in the prior art, and to provide a micro or nano vaccine system loaded with whole cell components of one or more cancer cells or tumor tissues for cross-prevention or cross-treatment of other vaccines. Approaches to different types of cancer.
  • the present invention adopts the following technical solutions: the application of nano cancer vaccine and/or micron cancer vaccine system based on whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs;
  • the cancer vaccine system of the invention comprises whole cell components, nano and/or micro particles; the whole cell components are cancer cell whole cell components and/or tumor tissue whole cell components.
  • the whole cell components are whole cell components of cancer cells and/or whole cell components of tumor tissues.
  • a vaccine system for cross-prevention or treatment of heterogeneous cancers including cancer cells or tumor tissue whole cell components, nanometers and/or microparticles, said vaccine system for cross-prevention or cross-treatment is different from the cancer cells used to prepare vaccines or other types of cancer of tumor tissue.
  • the invention is inventive in that the cancer for which the whole cell component is provided is different from the cancer for which prevention and treatment is desired.
  • the cancer that provides whole cell components and the cancer that needs prevention and treatment are all the same type of cancer, such as melanoma, lung cancer, breast cancer, etc.
  • the present invention proposes for the first time that the cancer that provides whole cell components and the cancer that needs prevention and treatment Cancers are different.
  • nano- and/or micro-particles loaded with whole cell components of melanoma are used to form nano-vaccine or micro-vaccine systems for the prevention and treatment of diseases such as lung cancer, breast cancer, and liver cancer.
  • the present invention can achieve very good results. The control effect is unpredictable.
  • the whole cell component-based nano cancer vaccine and/or micro cancer vaccine system further includes an immune enhancing adjuvant.
  • the whole cell component-based nano cancer vaccine and/or micro cancer vaccine system also includes an immune enhancing adjuvant in and/or on the surface.
  • the interior and/or surface of the particles further include an immune enhancing adjuvant.
  • the way of adding the immunoenhancing adjuvant includes loading in nanoparticles or microparticles, or loading on the surface of nanoparticles or microparticles, or loading in nanoparticles or microparticles and loading on the surface of nanoparticles or microparticles.
  • the immune-enhancing adjuvants include, but are not limited to, immune-enhancing agents derived from microorganisms, products of the human or animal immune system, innate immune stimulants, adaptive immune stimulants, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and other types of at least one category of .
  • the immune-enhancing adjuvants include but are not limited to pattern recognition receptor agonists, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyclonal anti-A, mineral Oil, virus-like particles, immune-enhanced reengineered influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG , immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium pumilus vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid , polyadenylic acid, alum,
  • the whole cell components for the preparation of cancer vaccines are derived from cancer cells and/or tumor tissues of one or more solid tumor cancers or non-solid tumor cancers;
  • the heterogeneous cancers for cross-prevention or cross-treatment are different Cancers of cancer cells or tumor tissues from which vaccines are prepared;
  • the heterogeneous cancers are one or more solid tumor cancers or non-solid tumor cancers.
  • cancer is solid tumor cancer or non-solid tumor cancer, such as endocrine system tumor, nervous system tumor, reproductive system tumor, digestive system tumor, urinary system tumor, immune system tumor, circulatory system tumor, respiratory system tumor, blood system tumor Tumors, tumors of the skin system.
  • Cancers are solid tumors or hematological system tumors, such as endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, urinary system tumors, immune system tumors, circulatory system tumors, respiratory system tumors, hematological system tumors, and skin system tumors.
  • the whole cell components are water-soluble components and/or water-insoluble components.
  • the whole cell component can be divided into two parts according to the solubility in pure water or an aqueous solution without a solubilizer: a water-soluble component and a water-insoluble component.
  • the water-soluble component is the original water-soluble part that is soluble in pure water or an aqueous solution without a solubilizer
  • the water-insoluble component is the original non-water-soluble part that is insoluble in pure water.
  • the part that is insoluble in water or an aqueous solution containing no solubilizing agent becomes soluble in an aqueous solution containing a solubilizing agent or an organic solvent.
  • Both the water-soluble part and the water-insoluble part in the whole cell fraction can be dissolved by a solubilizing aqueous solution containing a solubilizing agent or an organic solvent.
  • the solubilizer is at least one of the solubilizers that can increase the solubility of proteins or polypeptides in aqueous solution;
  • the organic solvent is an organic solvent that can dissolve proteins or polypeptides.
  • the solubilizer includes but not limited to urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerin, alkaline solution with pH greater than 7, acidic solution with pH less than 7, various protein degrading enzymes, albumin, lecithin, high Concentration Inorganic salt, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acid, glycoside, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin , lauryldimethylamine oxide, IGEPAL® CA-630.
  • the water-insoluble components can also be changed from insoluble in pure water to soluble by using other methods that can solubilize proteins and polypeptide fragments.
  • the organic solvent includes but not limited to DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, ethyl acetate.
  • the organic solvent can also use other organic solvent-containing methods that can solubilize proteins and polypeptide fragments.
  • whole cell components are loaded inside and/or on the surface of nanoparticles or microparticles, specifically, water-soluble components and/or water-insoluble components of whole cells are respectively or simultaneously loaded on nanoparticles and/or inside the micron particle, and/or separately or simultaneously loaded on the surface of the nanometer and/or micron particle.
  • the water-soluble components and/or water-insoluble components of the whole cells are separately or simultaneously loaded inside the particle, and/or separately or simultaneously loaded on the surface of the particle.
  • the loading method is that the water-soluble components and non-water-soluble components of the whole cell are separately or simultaneously loaded inside the particles, and/or are separately or simultaneously loaded on the surface of the particles; specifically, including but not limited to, the water-soluble components are simultaneously loaded on the Particles are neutralized and loaded on the surface of particles, water-insoluble components are loaded in particles and on the surface of particles at the same time, water-soluble components are loaded in particles but not water-soluble components are loaded on the surface of particles, water-insoluble components are loaded in particles and water-soluble The active ingredient is loaded on the particle surface, the water-soluble ingredient and the water-insoluble ingredient are loaded in the particle and only the water-insoluble ingredient is loaded on the particle surface, the water-soluble ingredient and the water-insoluble ingredient are loaded in the particle, and only the water-soluble ingredient is loaded on the particle On the surface, the water-soluble components are loaded in the particles, while the water-soluble components and the water-insoluble components are loaded on the particle surface at the same time, the water-insoluble components are loaded in the particles
  • the surface of the whole cell component-based cancer vaccine system may not be connected with a target head with active targeting function or may be connected with a target head with active targeting function.
  • the target head can lead the delivery system to target specific cells; the specific cells or tissues are dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils One or more of cells, basophils, lymph nodes, thymus, spleen, bone marrow.
  • the particle diameter of the nanoparticles is 1 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 100 nm to 600 nm; the particle diameter of the micron particles is 1 ⁇ m to 1000 ⁇ m, preferably 1 ⁇ m to 100 ⁇ m, more preferably 1 ⁇ m ⁇ 10 ⁇ m, more preferably 1 ⁇ m to 5 ⁇ m.
  • a cancer vaccine system based on whole cell components constructed from nanoparticles is a nano vaccine, and a cancer vaccine system based on whole cell components constructed from micron particles is called a micro vaccine.
  • the particle size of the nano vaccine is 1 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 100 nm to 600 nm; the particle size of the micron vaccine is 1 ⁇ m to 1000 ⁇ m, preferably 1 ⁇ m to 100 ⁇ m, more preferably 1 ⁇ m to 10 ⁇ m, more preferably 1 ⁇ m to 5 ⁇ m.
  • the surface of the nano-sized or micro-sized particles of the present invention can be neutral, negatively charged or positively charged.
  • the preparation materials of the nano and/or micro particles include but not limited to organic synthetic polymer materials, natural polymer materials or inorganic materials.
  • the organic synthetic polymer materials include but are not limited to PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, synthetic lipid, Synthetic nucleic acids;
  • the natural polymer materials include but not limited to lecithin, cholesterol, lipids, sodium alginate, protein, nucleic acid, gelatin, cell membrane components, starch, sugars, polypeptides;
  • the inorganic materials include but not Limited to ferric oxide, ferric oxide, calcium carbonate, calcium phosphate.
  • the shape of the nano and/or micron particles is any common shape
  • the shape of the prepared nano-vaccine or micro-vaccine is any common shape, including but not limited to spherical, ellipsoidal, barrel-shaped, polygonal, Rods, flakes, wires, worms, squares, triangles, butterflies or discs.
  • the cancer vaccine based on cancer cells or tumor tissue nanometers or micrometers disclosed by the present invention can cross-prevent many other different types of cancers.
  • the vaccine system loaded with whole cell components is used for cross-treatment or cross-prevention of various other cancers, Nano-sized or micron-sized particles and whole cell components loaded on the particles, or composed of nano-sized or micron-sized particles, whole cell components loaded on the particles, and an immune-enhancing adjuvant, said
  • the whole cell fraction refers to the water-soluble and/or water-insoluble components of whole cells in cancer cells or tumor tissues.
  • the present invention uses a cancer vaccine system derived from cancer cells and/or tumor tissue whole cell components for cross-prevention or cross-treatment of other different types of cancer, and the other types of cancer used for cross-prevention or cross-treatment can be one or more than one.
  • the nano-vaccine or micro-vaccine can be prepared according to the published preparation methods of nano-sized particles and micron-sized particles, including but not limited to common solvent evaporation method, dialysis method, extrusion method, and hot-melt method.
  • the nano-vaccine or micro-vaccine is prepared by the double emulsion method in the solvent evaporation method.
  • the vaccine system based on the whole cell components of cancer cells and/or tumor tissues of the present invention can simultaneously use nanoparticles or microparticles loaded with only water-soluble components and nanoparticle or microparticles loaded with only water-insoluble components when preventing or treating diseases.
  • the present invention provides a vaccine system that utilizes nanoscale or micronscale particles to deliver cell water-soluble components and/or water-insoluble components, and is used to prepare drugs for the prevention and treatment of non-same cancers .
  • the whole cell components of relevant cells or tissues are divided into two parts according to their solubility in pure water, the water-soluble part soluble in pure water and the insoluble part insoluble in pure water, and the water-soluble part and The water-insoluble part is loaded inside and/or on the surface of nanoparticles or microparticles, so most of the mutated proteins or polypeptides produced by cancer in cell components are loaded inside and/or on the surface of nanoparticles or microparticles .
  • the water-soluble part and the water-insoluble part in the cell component include the components of the whole cell; the water-soluble part and the water-insoluble part in the cell component can also be dissolved by the aqueous solution containing the solubilizer at the same time.
  • the unmutated proteins, polypeptides and genes that are the same as normal cell components will not cause immune response due to the immune tolerance produced during the development of the autoimmune system; while the mutations of genes, proteins and polypeptides produced by cancer, etc. will not cause immune responses due to the lack of autoimmunity Immune tolerance developed during phylogeny is thus immunogenic and can activate immune responses.
  • the use of these immunogenic substances produced by disease mutations in the whole cell components can be used for the treatment of cancer.
  • the broad-spectrum cancer vaccine system of the whole cell components of the present invention is used to prepare vaccines for cross-prevention and/or treatment of other different kinds of cancers.
  • the vaccine described in the present invention can be administered multiple times before or after the occurrence of cancer or after surgical resection of tumor tissue to activate the body's immune system, thereby delaying the progression of cancer, Treat cancer or prevent cancer from coming back.
  • Figures 1-8 are schematic structural diagrams of nano-sized particles or micron-sized vaccines loaded with water-soluble and water-insoluble cell components, wherein 1: water-soluble components in cell or tissue components; 2, cell or tissue components 3, the immune enhancing adjuvant; 4, nanoparticles or microparticles; 5: the core part of the nanoparticles.
  • Figure 9- Figure 19 is a schematic diagram of the structure of a nano-vaccine or a micro-vaccine loaded with water-soluble and water-insoluble cell components modified actively targeting the target head, wherein 1: the water-soluble component in the cell or tissue component; 2: Water-insoluble components in cells or tissue components; 3: immune adjuvants; 4: nanoparticles or microparticles; 5: the core part of nanoparticles; 6: targets that can target specific cells or tissues.
  • Figures 20-29 are the experimental results of tumor growth rate and survival period in mice when nano-vaccine or micro-vaccine prepared from one or more cancer tumor tissues in Examples 1-10 are used for cross-prevention or cross-treatment of other types of cancer; a, the experimental results of tumor growth rate when nano-vaccine or micro-vaccine cross-prevention or cross-treatment of other cancers (n ⁇ 8); b, the experimental results of mouse survival time when nano-vaccine or micro-vaccine cross-prevention or cross-treatment of 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, and the significant difference in figure b was analyzed by Kaplan-Meier and log- rank test analysis; ** indicates that this group has a significant difference with p ⁇ 0.005 compared with the PBS blank control group; ## indicates that this group has a significant difference with p ⁇ 0.005 compared with the blank nanoparticle + cell lysate control
  • Fig. 30 represents the preparation process and application field schematic diagram of the vaccine of the present invention
  • a water-soluble components and non-water-soluble components are respectively collected and prepared schematic diagrams of nano vaccines or micro vaccines
  • b adopting a solubilizing solution containing a solubilizing agent to dissolve Schematic diagram of whole cell components and preparation of nanovaccine or microvaccine.
  • the invention discloses a nano-vaccine or micro-vaccine system based on whole cell components of cancer cells and/or tumor tissues, and is used for cross-prevention or cross-treatment of other different types of cancers.
  • Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the methods and products of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • Invent technology is a nano-vaccine or micro-vaccine system based on whole cell components of cancer cells and/or tumor tissues, and is used for cross-prevention or cross-treatment of other different types of cancers.
  • the whole cell components of the nano or micro vaccine system based on cancer cells and/or tumor tissue whole cell components of the present invention are prepared from one or more cancer cells or tumor tissues, and used for cross-prevention or cross-treatment is different from preparation
  • the used cancer cells or other cancer types of tumor tissue can be one or more types of other cancers used for cross-prevention or cross-treatment.
  • the water-soluble components that are soluble in pure water or an aqueous solution without a solubilizing agent are firstly obtained, and then the water-insoluble components are dissolved by a solubilizing aqueous solution containing a solubilizing agent
  • the solubilization solution all the cell components can be converted into components that can be dissolved in the aqueous solution and then loaded inside and outside the nanoparticles or microparticles to prepare nano-vaccine or micro-vaccine for the prevention and treatment of cancer treat.
  • cells or tissues can also be lysed directly with a solubilizing aqueous solution containing a solubilizing agent to dissolve the whole cell components without collecting the water-soluble components and water-insoluble components separately, and the whole cell components dissolved in the solubilizing aqueous solution can be used
  • Cellular components make nanovaccine or microvaccine.
  • the present invention converts components insoluble in pure water or aqueous solutions without solubilizers in cells into soluble in specific solubilizing solutions and can be used to prepare nanoparticles and microparticles by using an aqueous solution containing a solubilizing agent, thereby improving The comprehensiveness and immunogenicity of the antigenic substances or components loaded by nano-vaccine or micro-vaccine.
  • the present invention divides the whole cell components in cancer cells and/or tumor tissues into water-soluble parts that can be dissolved in pure water or aqueous solutions without solubilizers and water-insoluble parts that can be dissolved in aqueous solutions with certain solubilizers, and
  • the water-soluble part and the water-insoluble part are entrapped in the nanoparticles or micro-particles and loaded on the surface thereof, thereby ensuring that most of the antigenic substances are loaded in the prepared vaccine.
  • the whole cell components of the present invention can be inactivated or (and) denatured before or (and) after lysis to prepare nano-vaccine or micro-vaccine, or can not be processed before or (and) after lysis Any inactivation or (and) denaturation treatment can directly prepare nano-vaccine or micro-vaccine.
  • the tumor tissue cells have been inactivated or (and) denatured before lysing. In actual use, they can also be inactivated or (and) denatured after cell lysis, or the cells can be lysed.
  • Inactivation or (and) denaturation treatment before and after lysis; the inactivation or (and) denaturation treatment method before or (and) after lysis of cells in some embodiments of the present invention is ultraviolet irradiation and high temperature heating. Inactivation or denaturation treatment methods such as radiation irradiation, high pressure, freeze-drying and formaldehyde can also be used in the process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific conditions.
  • FIG. 1-19 The structural schematic diagrams of the nano-vaccine or micro-vaccine system of the whole cell component of the present invention are shown in Figures 1-19. In actual use, only nanoparticles or microparticles of a specific structure may be used, or two or more nanoparticles or microparticles of different structures may be used simultaneously. In Fig. 1 and Fig. 2, the surface and interior of nanoparticles or microparticles all contain immune enhancing adjuvants; in Fig. 3-Fig.
  • Microparticles only contain immunoenhancing adjuvants on the outer surface;
  • Figure 7-8 have no immune enhancing adjuvants on the inner and outer surfaces of nanoparticles or microparticles;
  • Figure 1A, Figure 3A, Figure 5A and Figure 7A When the water-soluble or non-water-soluble components in the loaded cells or tissue components are distributed inside the nanoparticles or microparticles, no obvious inner core is formed;
  • Figure 1B, Figure 3B, Figure 5B and Figure 7B show that the nanoparticles or microparticles
  • the water-soluble or non-water-soluble components of the loaded cell or tissue components form a core part when they are distributed inside the nanoparticles or microparticles.
  • the core can be generated during the preparation process or formed by using polymers or inorganic salts, etc.
  • the water-soluble component or the non-water-soluble component in the cell or tissue component loaded by nanoparticle or microparticle form multiple inner cores when they are distributed inside the nanoparticle or microparticle
  • the inner core can be generated during the preparation process or formed by using polymers or inorganic salts
  • Figure 2B, Figure 4B, Figure 6B and Figure 8B The water-soluble components of cells or tissue contained in nanoparticles or microparticles When the active component or water-insoluble component is distributed inside the nanoparticle or microparticle, it is located in the outer layer of the formed inner core;
  • a The inside and surface of the nanoparticle or microparticle are all water-soluble components in the cell or tissue components ;
  • c Nanoparticles or microparticles contain water-insoluble components in cells or tissue components The components loaded on the surface are all water
  • FIG 9- Figure 10 the surface and interior of nanoparticles or microparticles contain immune adjuvants; in Figure 11- Figure 12, immune adjuvants are only distributed in the interior of nanoparticles or microparticles; Particles only contain immune adjuvant on the outer surface; Figure 15- Figure 16 has no immune adjuvant inside and outside the nanoparticle or microparticle; Figure 17 cell components and/or immune adjuvant are only distributed inside the nanoparticle or microparticle; Figure 18 Cell components and/or immune adjuvants are only distributed outside the nanoparticles or microparticles; Figure 19 Cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively.
  • the water-soluble or non-water-soluble components in the cells or tissue components are distributed in the outer layer of the inner core formed inside the nanoparticles or micro-particles; a: both the internal and surface loading of nanoparticles or micro-particles are cells or tissue groups the water-soluble components in the component; b: the nanoparticle or microparticle internally and surface-loaded are all water-insoluble components in the cell or tissue components; c: the nanoparticle or microparticle internally encapsulated is the cell or tissue
  • the water-insoluble components in the components are all water-soluble components in the cell or tissue components; d: the water-soluble components in the cells or tissue components are loaded on the surface of the nanoparticles or microparticles are the water-insoluble components in the cell or tissue components; e: the water-soluble components and the water-insoluble components in the cells or
  • the water-soluble or water-insoluble components of the cells or tissue components loaded by nanoparticles or microparticles in a, b and c do not form an obvious inner core when they are distributed inside the nanoparticles or microparticles ;
  • the water-soluble components or water-insoluble components in the cells or tissue components loaded by nanoparticles or microparticles in d, e and f are distributed in a core part inside the nanoparticles or microparticles; g, h and i
  • the water-soluble components or water-insoluble components in the cells or tissue components loaded by particles or microparticles are distributed in multiple core parts inside nanoparticles or microparticles; j, k and l are contained in nanoparticles or microparticles
  • the water-soluble or non-water-soluble components in the cell or tissue components are distributed in the outer layer of the inner core formed by nanoparticles or microparticles; a, d,
  • the method for preparing the vaccine system based on whole cell components described in the present invention is a common preparation method.
  • the double emulsion method in the solvent evaporation method is used to prepare nano or micro vaccines.
  • the nanoparticle preparation material used is organic polymer polylactic acid-glycolic acid copolymer (PLGA), and the immune adjuvant used is poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant, or CpG.
  • PLGA organic polymer polylactic acid-glycolic acid copolymer
  • the immune adjuvant used is poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant, or CpG.
  • the specific preparation method of the double-emulsion method used in the present invention is as follows: Step 1, adding a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of a medical solution containing a second predetermined concentration. In the organic phase of polymer materials.
  • the aqueous phase solution may contain the components in the cancer cell and/or tumor tissue lysate and the immunoenhancing adjuvant poly(I:C), BCG, manganese adjuvant or CpG; each component in the lysate
  • the components are respectively water-soluble components or original water-insoluble components dissolved in a solubilizing agent; or whole cell components dissolved in a solubilizing agent.
  • the predetermined concentration requires that the concentration of protein and peptide is greater than 1 ng/mL, enough antigen can be loaded to activate the relevant immune response.
  • the concentration of the immune enhancing adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
  • the aqueous phase solution contains each component in the cancer cell and/or tumor tissue lysate and the immunoenhancing adjuvant poly(I:C), BCG, manganese adjuvant or CpG; each group in the lysate
  • the components are respectively water-soluble components or original water-insoluble components dissolved in the solubilizing agent during preparation.
  • the concentration of the water-soluble components contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in the solubilizer, that is, the first predetermined concentration requires that the concentration of the protein polypeptide be greater than 1 ng/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 0.01 ng/mL.
  • the medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing a second predetermined concentration of the medical polymer material.
  • the medical polymer material is PLGA
  • the organic solvent is dichloromethane.
  • the range of the second predetermined concentration of the medical polymer material is 0.5mg/mL-5000mg/mL , preferably 100 mg/mL.
  • PLGA or modified PLGA is selected because the material is a biodegradable material and has been approved by the FDA as a drug excipient. Studies have shown that PLGA has a certain immune regulation function, so it is suitable as an auxiliary material for vaccine preparation.
  • the second predetermined volume of the organic phase is set according to its ratio with the first predetermined volume of the aqueous phase.
  • the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges 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 required to adjust the size of the prepared nanoparticles or microparticles.
  • step 2 the mixed solution obtained in step 1 is subjected to ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or homogenization treatment or microfluidic treatment.
  • This step is for nanometerization or micronization.
  • the length of ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles. Too long or too short will bring about changes in particle size. Therefore, it is necessary to select an appropriate ultrasonic time. .
  • the ultrasonic time is greater than 0.1 second, such as 2-200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm-500 rpm
  • the stirring time is greater than 1 minute, such as 60-600 seconds.
  • Step 3 adding the mixture obtained after the treatment in step 2 into a third predetermined volume of an aqueous solution containing an emulsifier of a third predetermined concentration and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or using micro Flow control processing.
  • the mixture obtained in step 2 is added to the emulsifier aqueous solution to continue ultrasonication or stirring or homogenization treatment or microfluidic treatment for nanometerization or micronization.
  • the emulsifier aqueous solution is 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 1:1.1 -1:1000 for setting, preferably 2:5.
  • the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time in this step the volume and concentration of the emulsifier aqueous solution are all based on the purpose of obtaining nanoparticles or microparticles of appropriate size.
  • Step 4 adding the liquid obtained after the treatment in step 3 into a fourth predetermined volume of an emulsifier aqueous solution of a fourth predetermined concentration, and performing stirring and/or vacuum treatment until predetermined conditions are met.
  • the emulsifier aqueous solution is still PVA
  • the fourth predetermined concentration is 5 mg/mL
  • the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of appropriate size.
  • the selection of the fourth predetermined volume is determined according to 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 between the third predetermined volume and the fourth predetermined volume can be adjusted.
  • the predetermined condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.
  • Step 5 after centrifuging the mixed solution that meets the predetermined conditions in step 4 at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in the fifth predetermined volume of the first Five predetermined concentrations of the aqueous solution containing the lyoprotectant or the 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), freeze-drying is not required, and subsequent nanoparticle or microparticle surface adsorption of cancer cells and/or Related experiments on tumor tissue lysates.
  • PBS physiological saline
  • the precipitate obtained in step 5 when the precipitate obtained in step 5 is resuspended in an aqueous solution containing a lyoprotectant, it needs to be lyophilized, and after lyophilization, the subsequent adsorption of cancer cells and/or tumors on the surface of nanoparticles or microparticles Related experiments on tissue lysates.
  • the lyoprotectant is selected from trehalose (Trehalose).
  • the fifth predetermined volume of the lyoprotectant in this step is 20 mL, and the fifth predetermined concentration is 4% by mass.
  • the reason for this setting is to not affect the effect of lyophilization in subsequent lyophilization. .
  • step 6 the suspension containing the lyoprotectant obtained in step 5 is lyophilized, and the lyophilized substance is used for future use.
  • Step 7 resuspend the sixth predetermined volume of the nanoparticle/microparticle-containing suspension obtained in step 5 in PBS (or normal saline) or resuspend with the sixth predetermined volume of PBS (or normal saline)
  • the freeze-dried freeze-dried substance containing nanoparticles/microparticles and freeze-drying protective agent obtained in step 6 is mixed with the seventh predetermined volume of water-soluble components or the original water-insoluble components dissolved in 8M urea.
  • Nano vaccines or micro vaccines are vaccine systems based on whole cell components.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferential volume ratio is 1:100 to 100:1, and the optimum volume ratio is 1:30 to 30:1 .
  • the resuspended nanoparticle or microparticle suspension has a volume of 10 mL, contains cancer cell lysate or contains water-soluble components in tumor tissue lysate or original non-alcohol dissolved in 8M urea.
  • the volume of the water-soluble component is 1 mL. The required volume and ratio of the two can be adjusted in the application.
  • the water-soluble components in the cancer cell and/or tumor tissue lysates or the original non-water-soluble components dissolved in 8M urea contain poly(I:C), Bacillus Calmette-Guerin (BCG), Manganese adjuvant or CpG, and the concentration of poly(I:C), BCG or CpG is greater than 1 ng/mL.
  • the particle size of nano-vaccine or micro-vaccine is nano-scale or micron-scale, which can ensure that the vaccine is phagocytized by antigen-presenting cells and activates the immune response. In order to improve the phagocytosis efficiency, the particle size should 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, most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • the particle size of the nanoparticle vaccine is 100nm-600nm
  • the particle size of the micron vaccine is 1 ⁇ m-5 ⁇ m.
  • urea and guanidine hydrochloride are used to solubilize the original water-insoluble components in the cancer cell and/or tumor tissue lysate, and any other components that can make the cancer cell and/or tumor
  • the original water-insoluble components in the tissue lysate are dissolved in the solubilizing substances of the aqueous solution, such as sodium deoxycholate, SDS, alkaline solution with pH greater than 7, acidic solution with pH less than 7, albumin, lecithin, high concentration Inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630; or the above-mentioned solub
  • 8M urea and 6M guanidine hydrochloride aqueous solution are used to solubilize the original water-insoluble components in cancer cells and/or tumor tissue lysates, and any other components that can make cancer cells
  • the original water-insoluble components in cell and/or tumor tissue lysates are dissolved in the urea concentration or guanidine hydrochloride concentration of the aqueous solution; sex component.
  • the preparation of nano-vaccine and micro-vaccine adopts the double emulsion method, but any other commonly used method for preparing nanoparticles or micro-particles can also be used in practice.
  • the preparation material of the nano-vaccine and the micro-vaccine is PLGA, but any other material capable of preparing nanoparticles or micro-particles can also be used in practice.
  • the water-soluble components in the cancer cell and/or tumor tissue lysates or the original water-insoluble components dissolved in 8M urea are respectively entrapped inside the nano/micro particles and adsorbed on the nano/micro particles.
  • the water-soluble components in cancer cell and/or tumor tissue lysates and the original water-insoluble components dissolved in 8M urea can also be mixed and then packed into the particles or adsorbed to the particles surface; or 8M urea can also be used to simultaneously dissolve the water-soluble component and the water-insoluble component and then entrapped inside the nanoparticle or microparticle and/or adsorbed on the surface of the nanoparticle or microparticle.
  • poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant and CpG are used as immune adjuvants.
  • Immune adjuvants such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanolic extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantibody A, mineral oil, virus-like particles, reconstitution for immune enhancement Influenza virion, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant poly ICLC, corynebacterium brevis vaccine, hemolytic chain Bacillus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum
  • the vaccines used in some embodiments are nano vaccines, and some embodiments use micro vaccines.
  • nano-vaccine or micro-vaccine according to the actual situation, that is, a vaccine system based on whole cell components of cancer cells and/or tumor tissues.
  • the vaccine system based on whole cell component of the present invention is composed of whole cell component, nano/micro particle, or composed of whole cell component, nano/micro particle and immune enhancing adjuvant.
  • the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. . Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
  • the methods used in the examples of the present invention are conventional methods; the materials and reagents used can be obtained from commercial sources.
  • the nanoparticle or microparticle structure, preparation method, and use strategy for disease treatment involved in the embodiments of the present invention are only representative methods, and the use of other nanoparticle or microparticle structures, preparation methods, and disease prevention or treatment
  • the strategy and the combination strategy with other drugs can also adopt the method described in the present invention.
  • the examples only list the application of the present invention in some cancers, but the present invention can also be used in any other types of cancer.
  • those skilled in the art can make conventional replacements based on the technical ideas of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention.
  • the specific administration time, administration frequency, administration regimen, and combination with other drugs can be adjusted according to the actual situation.
  • Example 1 The whole cell components of lung cancer tumor tissue are loaded inside and on the surface of nanoparticles for the prevention of melanoma: this example uses B16F10 mouse melanoma as a cancer model to illustrate how to prepare the whole cell components of lung cancer tumor tissue Nano-vaccine, and apply the vaccine to prevent melanoma. First, the tumor block of LLC lung cancer tissue is cracked to prepare water-soluble components and water-insoluble components of lung cancer tumor tissue.
  • the organic polymer material PLGA was used as the nanoparticle framework material, and Polyinosinic-polycytidylic acid (poly(I:C)) was used as the immune adjuvant to prepare nanoparticles loaded with water-soluble components and water-insoluble components by solvent evaporation method. vaccine.
  • the nanovaccine was then employed to prevent melanoma.
  • the component is the raw material source of the nano vaccine used to prevent melanoma prepared from melanoma tumor tissue.
  • the preparation of nano-vaccine and the blank nano-particles used as a control adopt the double emulsion method in the solvent evaporation method, and the molecular weight of the nano-particle preparation material PLGA used is 24KDa-38KDa.
  • the adjuvant is poly(I:C) And poly(I:C) is not only distributed inside the nanoparticles but also adsorbed on the surface of the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of nanoparticles is 320nm, and the average particle size of nano-vaccine after adsorption of cell components and immune adjuvant on the surface of nanoparticles is 340nm, and the surface potential of nano-vaccine is about -5mV .
  • Each 1mg PLGA nanoparticle is loaded with 180 ⁇ g protein or polypeptide component, and the poly(I:C) immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.01mg in total, and the inside and outside are divided in half.
  • the particle size of blank nanoparticles is 290nm.
  • Nano-vaccine for the treatment of cancer The control groups in this study were PBS group, blank nanoparticles + tumor tissue lysate group. Select 6-8-week-old female C57BL/6 as model mice to prepare melanoma-bearing mice.
  • the dosage regimen of the lung cancer tumor tissue nano-vaccine group is as follows: 200 ⁇ L of lung cancer tumor tissue lysates were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. 2 mg PLGA nanoparticles of water-soluble components and 200 ⁇ L of 2 mg PLGA nanoparticles loaded with original water-insoluble components dissolved in 8M urea in 200 ⁇ L; on day 0, 1.5 ⁇ 10 5 were subcutaneously inoculated in the lower right lower back of each mouse B16F10 cells.
  • the dosing regimen of the melanoma tumor tissue nanovaccine group is as follows: 200 ⁇ L of melanoma tumor tissue was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. 2 mg PLGA nanoparticles of the water-soluble component in the lysate and 200 ⁇ L of 2 mg PLGA nanoparticles of the original water-insoluble component dissolved in 8 M urea were loaded on the inside and on the surface; on day 0, 1.5 ⁇ 10 5 B16F10 cells.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was injected subcutaneously on the 49th day, 42th day, 35th day, 28th day and 14th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • Blank nanoparticles + tissue lysate control group 400 ⁇ L of blank nanoparticles and free lysate (equal to nanovaccine group) were subcutaneously injected 49 days, 42 days, 35 days, 28 days and 14 days before melanoma inoculation; Nanoparticles and free lysates were injected at different sites; on day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated on the lower right back of each mouse.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 2 The water-soluble components of 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 microparticles loaded with only the water-soluble components of LLC lung cancer cell components Micron vaccine, and the use of the vaccine to prevent melanoma.
  • LLC lung cancer cells were firstly lysed to prepare water-soluble components and water-insoluble components of LLC lung cancer cells. Then, the micron vaccine loaded with the water-soluble components of LLC cells is prepared by using the polymer material as the micron particle skeleton material and CpG as the immune adjuvant. And use the vaccine to prevent melanoma.
  • the LLC lung cancer cells were replaced with B16F10, and the water-soluble fraction derived from the melanoma cell lysate was prepared by the same method as above, which was the raw material source of the micron vaccine prepared by the melanoma cells.
  • micron vaccines In this example, the preparation of micron vaccines and the blank micron particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micron particle preparation material used is an organic polymer material PLGA with a molecular weight of 38KDa-54KDa , the immune adjuvant used is CpG, and CpG is not only distributed inside the microparticles but also adsorbed on the surface of the microparticles.
  • the preparation method is as described above.
  • the micron vaccine particle size obtained after adsorbing cell components and immune adjuvant on the surface of the micron particle is about 1.30 ⁇ m, and the average surface potential of the micron particle is about -5mV.
  • Each 1 mg of PLGA microparticles is loaded with 200 ⁇ g of protein or polypeptide components, and the CpG immune adjuvant used inside and outside of each 1 mg of PLGA microparticles is 0.01 mg, and the inside and outside are divided into half.
  • the particle size of the blank microparticles is about 1.25 ⁇ m, and the corresponding water-soluble components are replaced by pure water containing the same amount of CpG when the blank microparticles are prepared.
  • Micron vaccine for cancer prevention select 6-8 week old female C57BL/6 to prepare melanoma tumor-bearing mice.
  • the micro-vaccine group scheme is as follows: 400 ⁇ L of 4 mg PLGA micro-particles loaded with water-soluble components in cancer cell lysates were subcutaneously injected on the 28th, 21st, and 14th days before inoculation of melanoma, respectively. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS were subcutaneously injected on the 28th, 21st, and 14th days before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • Blank microparticles+cell lysate control group 400 ⁇ L of blank microparticles and cancer cell lysate equal to that in the vaccine were subcutaneously injected on the 28th day, 21st day, and 14th day before melanoma inoculation.
  • Example 3 Lung cancer tumor tissue lysate components loaded inside and on the surface of nanoparticles for the prevention of liver cancer: This example describes how to prepare a nano-vaccine loaded with lung cancer tumor tissue lysate components and apply the vaccine to prevent liver cancer Cross-prophylaxis against liver cancer using a vaccine prepared from lung cancer tumor tissue.
  • the lysed components of mouse LLC lung cancer tumor tissue were loaded inside and on the surface of nanoparticles to prepare nanovaccine.
  • the mouse LLC lung cancer tumor tissue was obtained and lysed to prepare the water-soluble fraction of the tumor tissue and the original water-insoluble fraction dissolved in 8M urea.
  • using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant to prepare nanovaccine loaded with water-soluble components and non-water-soluble components of the lysate, and use the vaccine to prevent Hepatocellular carcinoma 1-6 Liver cancer.
  • the nano-vaccine loaded with lung cancer tumor tissue is used for the prevention of liver cancer: select 6-8 week old female C57BL/6 to prepare Hepa 1-6 liver cancer tumor-bearing mice.
  • liver cancer cells On days 49, 42, 35, 28, and 14 before inoculation of liver cancer cells, 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in tissue lysates and 200 ⁇ L of internal and surface Both were loaded with 2mg of PLGA nanoparticles dissolved in 8M urea of the original water-insoluble component. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • Blank nanoparticles + free lysate control group Subcutaneously inject 400 ⁇ L of blank nanoparticles and the same amount of vaccine-loaded free lysate. Blank nanoparticles and free tissue lysates were injected at different sites. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 4 Whole cell components of lung cancer and melanoma tumor tissues loaded inside nanoparticles for the prevention of liver cancer: This example uses mouse liver cancer as a cancer model to illustrate how to prepare whole cell components loaded with lung cancer and melanoma tumor tissues Nano-vaccine, and apply the vaccine to prevent liver cancer.
  • lung cancer and melanoma tumor tissues were first lysed to prepare the water-soluble and water-insoluble fractions of the whole cell fraction. Then, using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant, a nanovaccine loaded with water-soluble components or water-insoluble components of lung cancer tumor mass and melanoma tumor mass was prepared by solvent evaporation method. , and use the nano-vaccine to prevent liver cancer.
  • the preparation of nano-vaccine adopts the double emulsion method in the solvent evaporation method
  • the molecular weight of the nanoparticle preparation material PLGA used is 7KDa-14KDa
  • the immune adjuvant used is poly(I:C) and poly(I:C) are distributed inside the nanoparticles.
  • the water-soluble component is a mixture (equal mass ratio) of the water-soluble component of lung cancer tumor tissue and the water-soluble component of melanoma tumor tissue
  • the water-insoluble component is the water-insoluble component of lung cancer tumor tissue and melanin A mixture of tumor tissue water-insoluble components (equal mass ratio).
  • the particle size of the nano-vaccine is about 300nm, and the average surface potential of the nano-particles is about -6mV.
  • Each 1 mg of PLGA nanoparticles is loaded with about 200 ⁇ g of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside of each 1 mg of PLGA nanoparticles is 0.01 mg.
  • the particle size of blank nanoparticles is about 240nm. When preparing blank nanoparticles, pure water containing poly(I:C) or 8M urea are used to replace the corresponding water-soluble components and non-water-soluble components. Blank nanoparticles are loaded with nano Vaccine equivalent poly(I:C).
  • the nano-vaccine is used for the prevention of cancer: the specific administration scheme and tumor growth monitoring scheme of the vaccine group and the control group are as in Example 3.
  • Example 5 Lysis components of melanoma tumor tissue and colon cancer tumor tissue are loaded inside and on the surface of nanoparticles for the treatment of pancreatic cancer:
  • This example uses mouse pancreatic cancer as a cancer model to illustrate how to prepare melanoma tumor tissue and colon cancer tumor tissue lysate components, and apply the vaccine to treat pancreatic cancer.
  • mouse B16F10 melanoma tumor tissue and MC38 colon cancer tumor tissue lysate components were loaded on the interior and surface of nanoparticles to prepare nanovaccine.
  • mouse melanoma and colon cancer tumor tissues were obtained and lysed to prepare the water-soluble fraction and the original water-insoluble fraction dissolved in 8M urea.
  • the water-soluble component is a 2:1 mass ratio mixture of the water-soluble component of the colon cancer tumor tissue and the water-soluble component of the melanoma tumor tissue; the water-insoluble component is the water-insoluble component of the colon cancer tumor tissue and a 2:1 mass ratio mixture of water-insoluble components of melanoma tumor tissue.
  • nanovaccine loaded with water-soluble and water-insoluble components of tumor tissue lysate was prepared. The vaccine was then used to treat Pan02 pancreatic cancer tumor-bearing mice.
  • Nano-vaccine for cancer treatment select 6-8 week old female C57BL/6 to prepare pancreatic cancer tumor mice. On day 0, 1 ⁇ 106 Pan02 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the vaccine group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in the lysate and 200 ⁇ L of lysate loaded with lysate on the 4th day, 7th day, 10th day, 15th day and 20th day, respectively. 2mg PLGA nanoparticles of the original water-insoluble component in 8M urea.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • Blank nanoparticles + lysate control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of free lysate loaded with the vaccine on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 6 Whole cell components of melanoma tumor tissue loaded inside microparticles for the prevention of lung cancer: This example uses mouse lung cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with whole cell components of melanoma tumor tissue, And apply the vaccine to prevent lung cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • mouse melanoma tumor tissues were obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea.
  • PLGA 50:50
  • mannose-modified PLGA were used as the microparticle framework material
  • CpG was used as the immune adjuvant to prepare the water-soluble and non-water-soluble components loaded with tumor tissue lysates by the solvent evaporation method.
  • micron vaccine The micron vaccine has the ability to target dendritic cells.
  • micron vaccines In this example, the micron vaccines and the empty micron particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micron particle preparation material PLGA (50:50) is used The molecular weight is 38KDa-54KDa, and the molecular weight of the mannose-modified PLGA (50:50) used is 38KDa-54KDa.
  • the mass ratio of unmodified PLGA to mannose-modified PLGA was 8:2.
  • the non-target modified micro-vaccine group was all prepared by unmodified PLGA.
  • the immune adjuvant used is CpG and the CpG is distributed inside the microparticles.
  • the preparation method is as mentioned above.
  • the average particle size of the micron particles is about 1.20 ⁇ m, and the average surface potential is about -8 mV. per 1 mg PLGA microparticles are loaded with 60 ⁇ g of protein or polypeptide components, and the CpG immune adjuvant used for each 1 mg of PLGA microparticles is 0.01 mg, and the inside and outside are divided in half.
  • the particle size of the blank microparticles is about 1.10 ⁇ m. When the blank microparticles are prepared, pure water or 8M urea containing the same amount of CpG is used to replace the corresponding water-soluble components and non-water-soluble components.
  • Micro-vaccine targeting dendritic cells for the prevention of cancer select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice.
  • the vaccine group received subcutaneous injections of 200 ⁇ L of 2 mg PLGA microparticles loaded with water-soluble components in cancer cell lysates and 200 ⁇ L of internal Both the surface and the surface are loaded with 2mg PLGA micron particles dissolved in 8M urea, which is the original water-insoluble component.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank microparticles + cell lysate control group were subcutaneously injected with 400 ⁇ L of blank microparticles and the same amount of free cells loaded with the vaccine on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. Lysate.
  • 2 ⁇ 10 6 LLC lung cancer cells were subcutaneously inoculated into the lower right lower back of each mouse. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 7 The whole cell components of lung cancer tumor tissue or melanoma tumor tissue are loaded inside and on the surface of nanoparticles, and the nano-vaccine with bacillus Calmette-Guerin (BCG) as an immune adjuvant is used for the prevention of liver cancer: this example takes mouse liver cancer as the cancer Model and use BCG as an immune adjuvant to illustrate how to use nano-vaccine loaded with whole cell components of lung cancer tumor tissue or melanoma tumor tissue to prevent liver cancer.
  • BCG bacillus Calmette-Guerin
  • the water-soluble and water-insoluble components of lung cancer or melanoma tumor tissue are lysed. Then, using PLGA as the nanoparticle framework material and BCG as the immune adjuvant to prepare nanovaccine loaded with water-soluble components and water-insoluble components of lung cancer or melanoma tumor tissue.
  • Nano-vaccine for the prevention of liver cancer Select female C57BL/6 as model mice to prepare Hepa 1-6 liver cancer tumor-bearing mice.
  • the vaccine group was subcutaneously injected with 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in tumor tissue lysate and 200 ⁇ L of the internal surface on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation, respectively. Both the surface and the surface are loaded with the 2mg PLGA nano-vaccine dissolved in the original water-insoluble component in 8M urea.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank nanoparticles + lysate control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of free lysate loaded with the vaccine on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. .
  • 2 ⁇ 106 Hepa1-6 liver cancer cells were subcutaneously inoculated into the armpit of each mouse. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 8 6M guanidine hydrochloride dissolves tumor tissue components of lung cancer and colon cancer and loads them inside and on the surface of microparticles for the treatment of breast cancer.
  • This example uses mouse breast cancer as a cancer model to illustrate how to use 6M guanidine hydrochloride to dissolve whole Cell components and preparation of micron vaccines loaded with whole cell components for the treatment of breast cancer.
  • 4T1 mouse triple-negative breast cancer cells were used as cancer cell models. Firstly, the tumor tissue cells of lung cancer and colon cancer were inactivated and denatured, and the tumor tissue was lysed with 6M guanidine hydrochloride to dissolve the whole cell components. Then, PLGA is used as the framework material of micron particles, and CpG is used as immune adjuvant to prepare the micron vaccine loaded with whole cell components. The microvaccine was then used to treat tumors in breast cancer-bearing mice.
  • the obtained tumor tissue cells were inactivated and denatured by ultraviolet light and high temperature heating respectively, and then the tumor tissue cells of lung cancer and colon cancer were lysed by appropriate amount of 6M guanidine hydrochloride and the tissue lysate was dissolved, and the tumor tissue lysate of lung cancer and colon cancer tumor tissue were lysed After the mixture is mixed, it is the source of raw materials for preparing vaccines.
  • micron vaccine and blank micron particles used PLGA (50:50) with a molecular weight of 38KD-54KD, and the preparation method was as described above.
  • CpG was used as an immune adjuvant.
  • the average particle size of the prepared micron vaccine is about 2.5 ⁇ m, and the surface potential of the micron particle is -4mV.
  • 210 ⁇ g of protein and polypeptide components are loaded inside and outside each 1 mg PLGA nanoparticle, and the CpG immune adjuvant used inside and outside each 1 mg PLGA nanoparticle is 0.01 mg in total, and the inside and outside are divided into half.
  • Micron vaccine for the treatment of cancer Select 6-8 weeks old female BALB/c to prepare 4T1 tumor-bearing mice. On day 0, 4 ⁇ 105 4T1 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the vaccine treatment group was subcutaneously injected with 400 ⁇ L of 4 mg PLGA micron vaccine loaded with whole cell components of tumor tissue inside and on the surface on the 4th, 7th, 10th, 15th and 20th days.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • Blank microparticles + lysate control group were subcutaneously injected with equal amount of tumor tissue lysate on the 4th day, 7th day, 10th day, 15th day and 20th day, and loaded with the same amount of CpG without any cell lysis 4mg PLGA blank micron particles of the material composition.
  • mice whose tumor volume exceeded 2000 mm3 were considered dead and were euthanized.
  • Example 9 Melanoma tumor tissue lysate components are loaded inside and on the surface of nanoparticles for the prevention of liver cancer: This example is based on how to prepare a nano-vaccine loaded with melanoma tumor tissue lysate components and apply the vaccine to prevent liver cancer To illustrate how to use a vaccine prepared from melanoma tumor tissue for cross-prevention of liver cancer.
  • mouse B16F10 melanoma tumor tissue lysate components were loaded on the inside and surface of nanoparticles to prepare nanovaccine.
  • mouse tumor tissue was obtained and lysed to prepare the water-soluble fraction of the tumor tissue and the original water-insoluble fraction dissolved in 6M guanidine hydrochloride.
  • poly(I:C) as an immune adjuvant or without an immune adjuvant to prepare nanovaccine loaded with water-soluble and water-insoluble components of the lysate, and using the Vaccine to prevent Hepa 1-6 liver cancer.
  • Nanovaccine loaded with tumor tissue is used for the prevention of liver cancer: 6-8 week old female C57BL/6 were selected to prepare Hepa 1-6 liver cancer tumor-bearing mice.
  • liver cancer cells On the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells, 200 ⁇ L of 2 mg PLGA nano-vaccine loaded with water-soluble components in tumor tissue lysates and 200 ⁇ L of internal and The surfaces are all loaded with 2 mg PLGA nano-vaccine dissolved in 8M urea, which is the original water-insoluble component.
  • 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • Blank nanoparticles + lysate control group Subcutaneously inject 400 ⁇ L of blank nanoparticles and free cell lysate. Blank nanoparticles and free cell lysates were injected at different sites. On day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • Example 10 Whole cell components of lung cancer cancer cells are loaded on the inside and surface of nanoparticles for the prevention of melanoma: This example illustrates how to prepare a nano-vaccine loaded with whole cell components of lung cancer cancer cells, and apply the vaccine to prevent melanoma tumor.
  • LLC cancer cells were first lysed to prepare the corresponding water-soluble components and water-insoluble components dissolved in 8M urea. Then, using PLGA as the framework material and colloidal manganese as the immune adjuvant to prepare the nano-vaccine.
  • nano-vaccine Preparation of nano-vaccine:
  • the nano-vaccine and the blank nano-particles were prepared by the double emulsion method, and the water-soluble components were loaded inside the nanoparticles instead of the water-soluble components on the surface of the nano-vaccine.
  • the nanoparticles used The molecular weight of the prepared material PLGA is 7KDa-17KDa, the immune adjuvant adopted is colloidal manganese and the colloidal manganese is distributed inside the nanoparticles.
  • the above samples were dissolved in 9 mL of PBS and mixed with 1 mL of non-water-soluble components (80 mg/mL) dissolved in 8M urea, and then used at room temperature for 10 min.
  • the average particle size of the nano-vaccine loaded with whole cell components is about 350nm, and the surface potential of the nano-vaccine is about -5mV; each 1 mg PLGA nano-particle is loaded with about 180 ⁇ g protein or polypeptide component.
  • the particle size of the blank nanoparticles is about 320nm, and the blank nanoparticles are loaded with an equal amount of colloidal manganese.
  • Nano-vaccine for the prevention of cancer mice in the vaccine group were given 400 ⁇ L of nano-vaccine containing 4 mg PLGA each time, and mice in the control group were given 400 ⁇ L of PBS or blank Nanoparticles + free lysate. Dosing regimen for prophylaxis and protocol for mouse tumor inoculation and monitoring Example 1.
  • the vaccine system with whole cell components of the present invention can be used to prepare drugs for cross-prevention and/or treatment of cancer, and its preparation process and application fields are shown in FIG. 30 .
  • the cells or tissues can be lysed, and then the water-soluble components and water-insoluble components can be collected separately to prepare nano-vaccine or micro-vaccine respectively; or directly use a solubilizing solution containing a solubilizing agent to directly lyse cells or tissues and dissolve whole cells Components and preparation of nano-vaccine or micro-vaccine.

Abstract

Provided is the application of a cancer vaccine system based on whole-cell components in the preparation of drugs for the cross-prevention or treatment of heterogeneous cancers, wherein the vaccine system uses nano-size or micro-size particles to deliver water-soluble components and/or non-water-soluble components of whole-cell components. As the water-soluble portion and/or the non-water-soluble portion are both loaded inside and/or on the surface of the nano-size or micro-size particles, variant proteins or polypeptides produced by cancers in the cell components are therefore also loaded inside and/or on the surface of the nano-size or micro-size particles. The immunogenic substances produced by mutation in the whole-cell components can be used for preventing and treating cancers. Therefore, the vaccine system based on whole cell components can prepare drugs for the cross-prevention and/or treatment of heterogeneous cancers.

Description

基于全细胞组分的癌症疫苗系统在制备交叉预防或治疗异种癌症药物中的应用Application of cancer vaccine system based on whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs 技术领域technical field
本发明属于免疫治疗领域，具体涉及一种基于癌细胞或肿瘤组织的广谱纳米或微米癌症疫苗，尤其是涉及一种基于一种或一种以上癌症的癌细胞和/或肿瘤组织的全细胞组分的广谱纳米或微米癌症疫苗及其在交叉预防多种其他不同类型的癌症中的应用。The invention belongs to the field of immunotherapy, in particular to a broad-spectrum nano or micro cancer vaccine based on cancer cells or tumor tissues, in particular to a whole cell based on cancer cells and/or tumor tissues of one or more cancers Components of broad-spectrum nano- or micro-cancer vaccines and their use in the cross-prophylaxis of many other different types of cancer.
背景技术Background technique
免疫是人体的一种生理功能，人体依靠这种功能识别“自己”和“非己”成分，从而破坏和排斥进入人体的抗原物质（如病毒和细菌等），或人体本身所产生的损伤细胞和肿瘤细胞等，以维持人体的健康。近些年来免疫技术的发展极其迅速，尤其是癌症的免疫治疗领域。随着对癌症认识的不断提高，人们发现人体的免疫系统和各类免疫细胞在抑制癌症发生、发展的过程中扮演着关键角色。通过调节机体免疫系统的强弱和平衡，有望影响和控制癌症的发生、发展和治疗。Immunity is a physiological function of the human body. The human body relies on this function to identify "self" and "non-self" components, thereby destroying and repelling antigenic substances (such as viruses and bacteria) entering the human body, or damaged cells produced by the human body itself and tumor cells to maintain human health. Immunotechnology has developed extremely rapidly in recent years, especially in the field of cancer immunotherapy. With the continuous improvement of the understanding of cancer, people have found that the human immune system and various immune cells play a key role in the process of inhibiting the occurrence and development of cancer. By regulating the strength and balance of the body's immune system, it is expected to affect and control the occurrence, development and treatment of cancer.
最近几年PD-1抗体和CAR-T等疗法相继获批进入临床，其临床效果良好，但是也有很大的局限性。癌症疫苗在癌症的预防和治疗中表现出了巨大的潜力。开发癌症疫苗的基础是选择合适的癌症抗原来激活人体免疫系统对异常突变的癌细胞的识别，而癌症细胞或者癌症肿瘤组织本身是最好的癌症抗原的来源。科学家曾采用新技术从癌症病人的肿瘤细胞分析鉴别癌症特异性的或癌症相关的抗原多肽，然后体外人工合成以制备癌症疫苗用于癌症的治疗。该技术在癌症病人的临床试验中表现出了一定的疗效，但是该类方法费时费力，花费巨大。而且所采用的方法都是只从癌细胞水溶性组分中提取分析癌细胞与正常细胞的差异进而寻找有差异的多肽，因而该类方法和技术只能找到有限的几种水溶性好的抗原多肽，从而极大的限制了该类方法的应用。而人体真实环境中的免疫原性强的抗原蛋白质或多肽很多都是在纯水中不溶的，需要借助与蛋白质结合、吸附或者位于膜上或膜表面以存在于体内，所以这部分不溶于纯水中的非水溶性蛋白质和多肽就非常重要和关键。所以将癌细胞或癌症组织全细胞组分作为疫苗用于预防和治疗癌症的疫苗的来源是很有前景的方法。现有技术公开了一种负载全细胞组分的靶向输送系统，为表面有靶头的纳米级尺寸或微米级尺寸的粒子，且所述粒子负载有癌细胞或肿瘤组织的全细胞组分；所述全细胞组分为细胞或组织中全细胞的水溶性成分和非水溶性成分，所述非水溶性成分通过增溶剂溶解；所述靶头与特定细胞或组织表面的分子结合进而帮助所述粒子进入细胞或组织；但是其公开的输送系统用于同种癌症的防治。现有技术未见关于采用一种或多种癌症组织或者细胞制备的癌症疫苗交叉预防和治疗不同癌症的报道。In recent years, therapies such as PD-1 antibody and CAR-T have been approved to enter the clinic one after another, and their clinical effects are good, but they also have great limitations. Cancer vaccines have shown great potential in the prevention and treatment of cancer. The basis for developing cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormally mutated cancer cells, and cancer cells or cancer tumor tissues themselves are the best source of cancer antigens. Scientists have used new technologies to analyze and identify cancer-specific or cancer-related antigenic polypeptides from tumor cells of cancer patients, and then artificially synthesize them in vitro to prepare cancer vaccines for cancer treatment. The technology has shown some efficacy in clinical trials of cancer patients, but such methods are time-consuming, laborious and costly. Moreover, the methods used only extract and analyze the differences between cancer cells and normal cells from the water-soluble components of cancer cells, and then look for the different polypeptides. Therefore, such methods and technologies can only find a limited number of antigens with good water solubility. Peptides, which greatly limit the application of this type of method. However, many antigenic proteins or polypeptides with strong immunogenicity in the real environment of the human body are insoluble in pure water. Water-insoluble proteins and peptides in water are very important and critical. Therefore, it is a promising approach to use cancer cells or whole cell components of cancer tissues as a source of vaccines for the prevention and treatment of cancer. The prior art discloses a targeted delivery system loaded with whole cell components, which is a nano-sized or micron-sized particle with a target head on the surface, and the particles are loaded with whole cell components of cancer cells or tumor tissues ; The whole cell component is the water-soluble component and the water-insoluble component of the whole cell in the cell or tissue, and the water-insoluble component is dissolved by a solubilizing agent; the target head is combined with molecules on the surface of a specific cell or tissue to help The particles enter cells or tissues; however, the disclosed delivery system is used for the prevention and treatment of the same cancer. In the prior art, there is no report on cross-prevention and treatment of different cancers using cancer vaccines prepared from one or more cancer tissues or cells.
技术问题technical problem
有鉴于此，本发明的目的在于针对现有技术存在的问题，提供一种负载一种或多种癌细胞或肿瘤组织的全细胞组分的微米或纳米疫苗系统用于交叉预防或交叉治疗其他不同种类癌症的方法。In view of this, the object of the present invention is to address the problems existing in the prior art, and to provide a micro or nano vaccine system loaded with whole cell components of one or more cancer cells or tumor tissues for cross-prevention or cross-treatment of other vaccines. Approaches to different types of cancer.
技术解决方案technical solution
为实现本发明的目的，本发明采用如下技术方案：基于全细胞组分的纳米癌症疫苗和/或微米癌症疫苗系统在制备交叉预防或治疗异种癌症药物中的应用；所述基于全细胞组分的癌症疫苗系统包括全细胞组分、纳米和/或微米粒子；所述全细胞组分为癌细胞全细胞组分和/或肿瘤组织全细胞组分。In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions: the application of nano cancer vaccine and/or micron cancer vaccine system based on whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs; The cancer vaccine system of the invention comprises whole cell components, nano and/or micro particles; the whole cell components are cancer cell whole cell components and/or tumor tissue whole cell components.
全细胞组分在制备交叉预防或治疗异种癌症药物中的应用；所述全细胞组分为癌细胞全细胞组分和/或肿瘤组织全细胞组分。Application of whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs; the whole cell components are whole cell components of cancer cells and/or whole cell components of tumor tissues.
一种交叉预防或治疗异种癌症的疫苗系统，包括癌细胞或肿瘤组织全细胞组分、纳米和/或微米粒子，所述疫苗系统用于交叉预防或交叉治疗不同于制备疫苗所使用的癌细胞或肿瘤组织的其他类型癌症。A vaccine system for cross-prevention or treatment of heterogeneous cancers, including cancer cells or tumor tissue whole cell components, nanometers and/or microparticles, said vaccine system for cross-prevention or cross-treatment is different from the cancer cells used to prepare vaccines or other types of cancer of tumor tissue.
本发明的创造性在于，提供全细胞组分的癌症与需要防治的癌症不同。现有技术中，提供全细胞组分的癌症与需要防治的癌症都是同种癌症，比如同为黑色素瘤、肺癌、乳腺癌等，本发明首次提出提供全细胞组分的癌症与需要防治的癌症不同，比如采用负载黑色素瘤的全细胞组分的纳米和/或微米粒子组成纳米疫苗或微米疫苗系统，用于肺癌、乳腺癌、肝癌等疾病的防治，实验证实，本发明可以取得非常好的防治效果，具有预料不到性。The invention is inventive in that the cancer for which the whole cell component is provided is different from the cancer for which prevention and treatment is desired. In the prior art, the cancer that provides whole cell components and the cancer that needs prevention and treatment are all the same type of cancer, such as melanoma, lung cancer, breast cancer, etc. The present invention proposes for the first time that the cancer that provides whole cell components and the cancer that needs prevention and treatment Cancers are different. For example, nano- and/or micro-particles loaded with whole cell components of melanoma are used to form nano-vaccine or micro-vaccine systems for the prevention and treatment of diseases such as lung cancer, breast cancer, and liver cancer. Experiments have proved that the present invention can achieve very good results. The control effect is unpredictable.
本发明中，所述基于全细胞组分的纳米癌症疫苗和/或微米癌症疫苗系统还包括免疫增强佐剂。具体的，所述基于全细胞组分的纳米癌症疫苗和/或微米癌症疫苗系统内部和/或表面还包括免疫增强佐剂。在一些实施方案中，上述基于全细胞组分的癌症疫苗系统中，所述粒子内部和/或表面还包括免疫增强佐剂。所述的免疫增强佐剂的添加方式包括装载于纳米粒子或微米粒子内，或者负载于纳米粒子或微米粒子表面，或者同时装载于纳米粒子或微米粒子内及负载于纳米粒子或微米粒子表面。所述免疫增强佐剂包括但不限于微生物来源的免疫增强剂、人或动物免疫系统的产物、固有免疫激动剂、适应性免疫激动剂、化学合成药物、真菌多糖类、中药及其他类中的至少一类。所述免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗（BCG）、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂CpG、免疫佐剂poly(I:C)、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、植物油、内毒素、脂质体佐剂、GM-CSF、MF59、双链RNA、双链DNA、铝佐剂、锰佐剂、CAF01、人参、黄芪的有效成分中的至少一种。本领域技术人员可以理解，所述免疫增强佐剂也可采用其他可使免疫反应增强的物质。In the present invention, the whole cell component-based nano cancer vaccine and/or micro cancer vaccine system further includes an immune enhancing adjuvant. Specifically, the whole cell component-based nano cancer vaccine and/or micro cancer vaccine system also includes an immune enhancing adjuvant in and/or on the surface. In some embodiments, in the above cancer vaccine system based on whole cell components, the interior and/or surface of the particles further include an immune enhancing adjuvant. The way of adding the immunoenhancing adjuvant includes loading in nanoparticles or microparticles, or loading on the surface of nanoparticles or microparticles, or loading in nanoparticles or microparticles and loading on the surface of nanoparticles or microparticles. The immune-enhancing adjuvants include, but are not limited to, immune-enhancing agents derived from microorganisms, products of the human or animal immune system, innate immune stimulants, adaptive immune stimulants, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and other types of at least one category of . The immune-enhancing adjuvants include but are not limited to pattern recognition receptor agonists, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyclonal anti-A, mineral Oil, virus-like particles, immune-enhanced reengineered influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin, immune adjuvant CpG , immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium pumilus vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid , polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, double-stranded RNA, double-stranded DNA, aluminum adjuvant, manganese adjuvant, CAF01, ginseng , at least one of the active ingredients of Radix Astragali. Those skilled in the art can understand that the immune enhancing adjuvant can also use other substances that can enhance the immune response.
本发明中，所述制备癌症疫苗的全细胞组分来源于一种或者多种实体瘤癌症或者非实体瘤癌症的癌细胞和/或肿瘤组织；所述交叉预防或交叉治疗的异种癌症为不同于制备疫苗的癌细胞或肿瘤组织的癌症；所述异种癌症为一种或多种实体瘤癌症或者非实体瘤癌症。In the present invention, the whole cell components for the preparation of cancer vaccines are derived from cancer cells and/or tumor tissues of one or more solid tumor cancers or non-solid tumor cancers; the heterogeneous cancers for cross-prevention or cross-treatment are different Cancers of cancer cells or tumor tissues from which vaccines are prepared; the heterogeneous cancers are one or more solid tumor cancers or non-solid tumor cancers.
本发明中，癌症为实体瘤癌症或者非实体瘤癌症，比如内分泌系统肿瘤、神经系统肿瘤、生殖系统肿瘤、消化系统肿瘤、泌尿系统肿瘤、免疫系统肿瘤、循环系统肿瘤、呼吸系统肿瘤、血液系统肿瘤、皮肤系统肿瘤。癌症为实体瘤或者血液系统肿瘤，比如内分泌系统肿瘤、神经系统肿瘤、生殖系统肿瘤、消化系统肿瘤、泌尿系统肿瘤、免疫系统肿瘤、循环系统肿瘤、呼吸系统肿瘤、血液系统肿瘤、皮肤系统肿瘤。In the present invention, cancer is solid tumor cancer or non-solid tumor cancer, such as endocrine system tumor, nervous system tumor, reproductive system tumor, digestive system tumor, urinary system tumor, immune system tumor, circulatory system tumor, respiratory system tumor, blood system tumor Tumors, tumors of the skin system. Cancers are solid tumors or hematological system tumors, such as endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, urinary system tumors, immune system tumors, circulatory system tumors, respiratory system tumors, hematological system tumors, and skin system tumors.
本发明中，全细胞组分为水溶性成份和/或非水溶性成份。所述全细胞组分按照在纯水或不含增溶剂的水溶液中的溶解性可分为两部分：水溶性成分和非水溶性成分。所述水溶性成分为可溶于纯水或不含增溶剂的水溶液的原水溶性部分，所述非水溶性成分为在纯水中不溶的原非水溶性部分，采用适当增溶方法由在纯水或不含增溶剂的水溶液中不溶变为在含增溶剂的水溶液中或有机溶剂中可溶的部分。所述全细胞组分中的水溶性部分和非水溶性部分都可以被含增溶剂的增溶水溶液或有机溶剂溶解。所述增溶剂为可以增加蛋白质或多肽在水溶液中溶解性的增溶剂中的至少一种；所述有机溶剂为可以溶解蛋白质或多肽的有机溶剂。所述增溶剂包括但不限于尿素、盐酸胍、脱氧胆酸钠、SDS、甘油、pH大于7的碱性溶液、pH小于7的酸性溶液、各类蛋白质降解酶、白蛋白、卵磷脂、高浓度无机盐、Triton、吐温、DMSO、乙腈、乙醇、甲醇、DMF、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、IGEPAL®CA-630。本领域技术人员可以理解，所述非水溶性成分也可采用其他可使蛋白质和多肽片段增溶的方法由在纯水中不溶变为可溶。所述有机溶剂包括但不限于DMSO、乙腈、乙醇、甲醇、DMF、异丙醇、丙醇、二氯甲烷、乙酸乙酯。本领域技术人员可以理解，所述有机溶剂也可采用其他可使蛋白质和多肽片段增溶的含有机溶剂的方法。 In the present invention, the whole cell components are water-soluble components and/or water-insoluble components. The whole cell component can be divided into two parts according to the solubility in pure water or an aqueous solution without a solubilizer: a water-soluble component and a water-insoluble component. The water-soluble component is the original water-soluble part that is soluble in pure water or an aqueous solution without a solubilizer, and the water-insoluble component is the original non-water-soluble part that is insoluble in pure water. The part that is insoluble in water or an aqueous solution containing no solubilizing agent becomes soluble in an aqueous solution containing a solubilizing agent or an organic solvent. Both the water-soluble part and the water-insoluble part in the whole cell fraction can be dissolved by a solubilizing aqueous solution containing a solubilizing agent or an organic solvent. The solubilizer is at least one of the solubilizers that can increase the solubility of proteins or polypeptides in aqueous solution; the organic solvent is an organic solvent that can dissolve proteins or polypeptides. The solubilizer includes but not limited to urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerin, alkaline solution with pH greater than 7, acidic solution with pH less than 7, various protein degrading enzymes, albumin, lecithin, high Concentration Inorganic salt, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acid, glycoside, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin , lauryldimethylamine oxide, IGEPAL® CA-630. Those skilled in the art can understand that the water-insoluble components can also be changed from insoluble in pure water to soluble by using other methods that can solubilize proteins and polypeptide fragments. The organic solvent includes but not limited to DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, ethyl acetate. Those skilled in the art can understand that the organic solvent can also use other organic solvent-containing methods that can solubilize proteins and polypeptide fragments.
本发明中，全细胞组分被负载于纳米粒子或微米粒子的内部和/或表面，具体的，全细胞的水溶性成分和/或非水溶性成分分别或同时被包载于纳米和/或微米粒子内部，和/或分别或同时负载于纳米和/或微米粒子表面。全细胞的水溶性成分和/或非水溶性成分分别或同时被包载于粒子内部，和/或分别或同时负载于粒子表面。所述负载方式为全细胞的水溶性成分和非水溶性成分分别或同时被包载于粒子内部，和/或分别或同时负载于粒子表面；具体的，包括但不仅限于水溶性成分同时装载于粒子中和负载于粒子表面，非水溶性成分同时装载于粒子中和负载于粒子表面，水溶性成分装载于粒子中而非水溶性成分负载于粒子表面，非水溶性成分装载于粒子中而水溶性成分负载于粒子表面，水溶性成分和非水溶性成分装载于粒子中而只有非水溶性成分负载于粒子表面，水溶性成分和非水溶性成分装载于粒子中而只有水溶性成分负载于粒子表面，水溶性成分装载于粒子中而水溶性成分和非水溶性成分同时负载于粒子表面，非水溶性成分装载于粒子中而水溶性成分和非水溶性成分同时负载于粒子表面，水溶性成分和非水溶性成分同时装载于粒子中而且水溶性成分和非水溶性成分同时负载于粒子表面。In the present invention, whole cell components are loaded inside and/or on the surface of nanoparticles or microparticles, specifically, water-soluble components and/or water-insoluble components of whole cells are respectively or simultaneously loaded on nanoparticles and/or inside the micron particle, and/or separately or simultaneously loaded on the surface of the nanometer and/or micron particle. The water-soluble components and/or water-insoluble components of the whole cells are separately or simultaneously loaded inside the particle, and/or separately or simultaneously loaded on the surface of the particle. The loading method is that the water-soluble components and non-water-soluble components of the whole cell are separately or simultaneously loaded inside the particles, and/or are separately or simultaneously loaded on the surface of the particles; specifically, including but not limited to, the water-soluble components are simultaneously loaded on the Particles are neutralized and loaded on the surface of particles, water-insoluble components are loaded in particles and on the surface of particles at the same time, water-soluble components are loaded in particles but not water-soluble components are loaded on the surface of particles, water-insoluble components are loaded in particles and water-soluble The active ingredient is loaded on the particle surface, the water-soluble ingredient and the water-insoluble ingredient are loaded in the particle and only the water-insoluble ingredient is loaded on the particle surface, the water-soluble ingredient and the water-insoluble ingredient are loaded in the particle, and only the water-soluble ingredient is loaded on the particle On the surface, the water-soluble components are loaded in the particles, while the water-soluble components and the water-insoluble components are loaded on the particle surface at the same time, the water-insoluble components are loaded in the particles, and the water-soluble components and the water-insoluble The water-soluble and water-insoluble components are simultaneously loaded in the particles and the water-soluble and water-insoluble components are simultaneously loaded on the surface of the particles.
本发明中，所述基于全细胞组分的癌症疫苗系统表面可以不连接具有主动靶向功能的靶头或者连接有主动靶向功能的靶头。所述靶头可带领输送系统靶向到特定细胞；所述特定细胞或组织为树突状细胞、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、骨髓中的一种或两种以上。In the present invention, the surface of the whole cell component-based cancer vaccine system may not be connected with a target head with active targeting function or may be connected with a target head with active targeting function. The target head can lead the delivery system to target specific cells; the specific cells or tissues are dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils One or more of cells, basophils, lymph nodes, thymus, spleen, bone marrow.
本发明中，所述纳米粒子的粒径为1nm～1000nm，优选为50nm～800nm，进一步优选为100nm～600nm；所述微米粒子的粒径为1μm～1000μm，优选为1μm～100μm进一步优选为1μm～10μm，更优选为1μm～5μm。由纳米粒子构建的基于全细胞组分的癌症疫苗系统为纳米疫苗，由微米粒子构建的基于全细胞组分的癌症疫苗系统为微米疫苗。进一步的，所述纳米疫苗的粒径为1nm～1000nm，优选为50nm～800nm，进一步优选为100nm～600nm；所述微米疫苗的粒径为1μm～1000μm，优选为1μm～100μm进一步优选为1μm～10μm，更优选为1μm～5μm。本发明的纳米尺寸粒子或微米尺寸粒子表面可为电中性，带负电或者带正电。In the present invention, the particle diameter of the nanoparticles is 1 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 100 nm to 600 nm; the particle diameter of the micron particles is 1 μm to 1000 μm, preferably 1 μm to 100 μm, more preferably 1 μm ~10 μm, more preferably 1 μm to 5 μm. A cancer vaccine system based on whole cell components constructed from nanoparticles is a nano vaccine, and a cancer vaccine system based on whole cell components constructed from micron particles is called a micro vaccine. Further, the particle size of the nano vaccine is 1 nm to 1000 nm, preferably 50 nm to 800 nm, more preferably 100 nm to 600 nm; the particle size of the micron vaccine is 1 μm to 1000 μm, preferably 1 μm to 100 μm, more preferably 1 μm to 10 μm, more preferably 1 μm to 5 μm. The surface of the nano-sized or micro-sized particles of the present invention can be neutral, negatively charged or positively charged.
本发明中，所述纳米和/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。所述有机合成高分子材料包括但不限于PLGA、PLA、PGA、PEG、PCL、Poloxamer、PVA、PVP、PEI、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽、合成脂质、合成核酸；所述的天然高分子材料包括但不限于卵磷脂、胆固醇、脂类、海藻酸钠、蛋白质、核酸、明胶、细胞膜成分、淀粉、糖类、多肽；所述的无机材料包括但不限于三氧化二铁、四氧化三铁、碳酸钙、磷酸钙。In the present invention, the preparation materials of the nano and/or micro particles include but not limited to organic synthetic polymer materials, natural polymer materials or inorganic materials. The organic synthetic polymer materials include but are not limited to PLGA, PLA, PGA, PEG, PCL, Poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, synthetic lipid, Synthetic nucleic acids; the natural polymer materials include but not limited to lecithin, cholesterol, lipids, sodium alginate, protein, nucleic acid, gelatin, cell membrane components, starch, sugars, polypeptides; the inorganic materials include but not Limited to ferric oxide, ferric oxide, calcium carbonate, calcium phosphate.
本发明中，所述纳米和/或微米粒子的形状为常见的任意形状，制备的纳米疫苗或微米疫苗的形状为常见的任意形状，包括但不限于球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形。In the present invention, the shape of the nano and/or micron particles is any common shape, and the shape of the prepared nano-vaccine or micro-vaccine is any common shape, including but not limited to spherical, ellipsoidal, barrel-shaped, polygonal, Rods, flakes, wires, worms, squares, triangles, butterflies or discs.
本发明公开的基于癌细胞或肿瘤组织纳米或微米的癌症疫苗可以交叉预防多种其他不同类型的癌症，该负载全细胞组分的疫苗系统用于交叉治疗或交叉预防多种其他癌症时，由纳米级尺寸或微米级尺寸的粒子和所述粒子负载的全细胞组分组成或者由纳米级尺寸或微米级尺寸的粒子和所述粒子负载的全细胞组分、免疫增强佐剂组成，所述全细胞组分为癌细胞或肿瘤组织中全细胞的水溶性成分和/或非水溶性成分。The cancer vaccine based on cancer cells or tumor tissue nanometers or micrometers disclosed by the present invention can cross-prevent many other different types of cancers. When the vaccine system loaded with whole cell components is used for cross-treatment or cross-prevention of various other cancers, Nano-sized or micron-sized particles and whole cell components loaded on the particles, or composed of nano-sized or micron-sized particles, whole cell components loaded on the particles, and an immune-enhancing adjuvant, said The whole cell fraction refers to the water-soluble and/or water-insoluble components of whole cells in cancer cells or tumor tissues.
本发明首次将来自于癌细胞和/或肿瘤组织全细胞组分的癌症疫苗系统用于交叉预防或交叉治疗其他不同种类的癌症，用于交叉预防或交叉治疗的其他癌症类型可为一种或一种以上。For the first time, the present invention uses a cancer vaccine system derived from cancer cells and/or tumor tissue whole cell components for cross-prevention or cross-treatment of other different types of cancer, and the other types of cancer used for cross-prevention or cross-treatment can be one or more than one.
本发明中，所述纳米疫苗或微米疫苗可以按照纳米尺寸粒子和微米尺寸粒子已公开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。在一些实施方案中，所述纳米疫苗或微米疫苗采用溶剂挥发法中的复乳法制备得到。In the present invention, the nano-vaccine or micro-vaccine can be prepared according to the published preparation methods of nano-sized particles and micron-sized particles, including but not limited to common solvent evaporation method, dialysis method, extrusion method, and hot-melt method. In some embodiments, the nano-vaccine or micro-vaccine is prepared by the double emulsion method in the solvent evaporation method.
本发明所述基于癌细胞和/或肿瘤组织的全细胞组分的疫苗系统在预防或治疗疾病时可以同时使用只负载水溶性组分的纳米粒子或微米粒子和只负载非水溶性组分的纳米粒子或微米粒子、使用只负载水溶性组分的纳米粒子或微米粒子、使用只负载非水溶性组分的纳米粒子或微米粒子或者使用同时负载水溶性组分和非水溶性组分的纳米粒子或微米粒子。The vaccine system based on the whole cell components of cancer cells and/or tumor tissues of the present invention can simultaneously use nanoparticles or microparticles loaded with only water-soluble components and nanoparticle or microparticles loaded with only water-insoluble components when preventing or treating diseases. Nanoparticles or microparticles, using nanoparticles or microparticles loaded with only water-soluble components, using nanoparticles or microparticles loaded with only water-insoluble components, or using nanoparticles loaded with both water-soluble components and water-insoluble components particles or microparticles.
由上述技术方案可知本发明提供了一种利用纳米级尺寸或微米级尺寸的粒子递送细胞水溶性成分和/或非水溶性成分的疫苗系统，以及用于制备预防和治疗非同种癌症的药物。因为相关细胞或组织的全细胞组分按照在纯水中的溶解性被分为两部分，可溶于纯水的水溶性部分和在纯水中不溶的非水溶性部分，并且水溶性部分和非水溶性部分都被负载于纳米粒子或微米粒子内部和/或表面，所以细胞组分中因为癌症所产生的变异蛋白质或多肽就大部分都被负载于纳米粒子或微米粒子内部和/或表面。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；细胞组分中水溶性部分和非水溶性部分也可以同时被含有增溶剂的水溶液溶解。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；而因为癌症等产生的基因、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免疫原性的物质即可用于癌症的治疗。From the above technical scheme, it can be known that the present invention provides a vaccine system that utilizes nanoscale or micronscale particles to deliver cell water-soluble components and/or water-insoluble components, and is used to prepare drugs for the prevention and treatment of non-same cancers . Because the whole cell components of relevant cells or tissues are divided into two parts according to their solubility in pure water, the water-soluble part soluble in pure water and the insoluble part insoluble in pure water, and the water-soluble part and The water-insoluble part is loaded inside and/or on the surface of nanoparticles or microparticles, so most of the mutated proteins or polypeptides produced by cancer in cell components are loaded inside and/or on the surface of nanoparticles or microparticles . The water-soluble part and the water-insoluble part in the cell component include the components of the whole cell; the water-soluble part and the water-insoluble part in the cell component can also be dissolved by the aqueous solution containing the solubilizer at the same time. Among them, the unmutated proteins, polypeptides and genes that are the same as normal cell components will not cause immune response due to the immune tolerance produced during the development of the autoimmune system; while the mutations of genes, proteins and polypeptides produced by cancer, etc. will not cause immune responses due to the lack of autoimmunity Immune tolerance developed during phylogeny is thus immunogenic and can activate immune responses. The use of these immunogenic substances produced by disease mutations in the whole cell components can be used for the treatment of cancer.
有益效果Beneficial effect
本发明所述全细胞组分的广谱癌症疫苗系统用于制备交叉预防和/或治疗其他不同种类癌症的疫苗。在用作癌症疫苗以交叉预防和治疗癌症时，本发明所述的疫苗可以在癌症发生前或癌症发生后或手术切除肿瘤组织后多次给药以激活机体免疫系统，从而延缓癌症的进展、治疗癌症或者预防癌症的复发。The broad-spectrum cancer vaccine system of the whole cell components of the present invention is used to prepare vaccines for cross-prevention and/or treatment of other different kinds of cancers. When used as a cancer vaccine for cross-prevention and treatment of cancer, the vaccine described in the present invention can be administered multiple times before or after the occurrence of cancer or after surgical resection of tumor tissue to activate the body's immune system, thereby delaying the progression of cancer, Treat cancer or prevent cancer from coming back.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.
图1-图8为载有水溶性和非水溶性细胞组分的纳米尺寸粒子或微米尺寸疫苗的结构示意图，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，免疫增强佐剂；4，纳米粒子或微米粒子；5：纳米粒子中的内核部分。Figures 1-8 are schematic structural diagrams of nano-sized particles or micron-sized vaccines loaded with water-soluble and water-insoluble cell components, wherein 1: water-soluble components in cell or tissue components; 2, cell or tissue components 3, the immune enhancing adjuvant; 4, nanoparticles or microparticles; 5: the core part of the nanoparticles.
图9-图19为主动靶向靶头修饰的载有水溶性和非水溶性细胞组分的纳米疫苗或微米疫苗的结构示意图，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。Figure 9-Figure 19 is a schematic diagram of the structure of a nano-vaccine or a micro-vaccine loaded with water-soluble and water-insoluble cell components modified actively targeting the target head, wherein 1: the water-soluble component in the cell or tissue component; 2: Water-insoluble components in cells or tissue components; 3: immune adjuvants; 4: nanoparticles or microparticles; 5: the core part of nanoparticles; 6: targets that can target specific cells or tissues.
图20-29分别为实施例1-10中用一种或多种癌症肿瘤组织制备的纳米疫苗或微米疫苗用于交叉预防或交叉治疗其他种类癌症时小鼠肿瘤生长速度和生存期实验结果；a, 纳米疫苗或微米疫苗交叉预防或交叉治疗其他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交叉预防或交叉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）；a图中肿瘤生长抑制实验的显著性差异采用ANOVA法分析，b图中显著性差异采用Kaplan-Meier和log-rank test 分析；**表示该组与PBS空白对照组相比p＜0.005，有显著性差异；##代表该组与空白纳米粒+细胞裂解物对照组相比p＜0.005，有显著性差异；***表示该组与PBS空白对照组相比p＜0.0005，有显著性差异；###代表该组与空白纳米粒+细胞裂解物对照组相比p＜0.0005，有显著性差异。Figures 20-29 are the experimental results of tumor growth rate and survival period in mice when nano-vaccine or micro-vaccine prepared from one or more cancer tumor tissues in Examples 1-10 are used for cross-prevention or cross-treatment of other types of cancer; a, the experimental results of tumor growth rate when nano-vaccine or micro-vaccine cross-prevention or cross-treatment of other cancers (n≥8); b, the experimental results of mouse survival time when nano-vaccine or micro-vaccine cross-prevention or cross-treatment of 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, and the significant difference in figure b was analyzed by Kaplan-Meier and log- rank test analysis; ** indicates that this group has a significant difference with p<0.005 compared with the PBS blank control group; ## indicates that this group has a significant difference with p<0.005 compared with the blank nanoparticle + cell lysate control group; *** indicates that this group has a significant difference with p<0.0005 compared with the PBS blank control group; ### indicates that this group has a significant difference with p<0.0005 compared with the blank nanoparticle + cell lysate control group.
图30表示本发明所述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:采用含有增溶剂的增溶液溶解全细胞组分和制备纳米疫苗或微米疫苗的示意图。Fig. 30 represents the preparation process and application field schematic diagram of the vaccine of the present invention; a: water-soluble components and non-water-soluble components are respectively collected and prepared schematic diagrams of nano vaccines or micro vaccines; b: adopting a solubilizing solution containing a solubilizing agent to dissolve Schematic diagram of whole cell components and preparation of nanovaccine or microvaccine.
本发明的实施方式Embodiments of the present invention
本发明公开了一种基于癌细胞和/或肿瘤组织全细胞组分的纳米疫苗或微米疫苗系统并应用交叉预防或交叉治疗其他不同类型的癌症。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a nano-vaccine or micro-vaccine system based on whole cell components of cancer cells and/or tumor tissues, and is used for cross-prevention or cross-treatment of other different types of cancers. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and products of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.
本发明基于癌细胞和/或肿瘤组织全细胞组分的纳米或微米疫苗系统的全细胞组分由一种或一种以上的癌细胞或肿瘤组织制备，用于交叉预防或交叉治疗不同于制备所使用的癌细胞或肿瘤组织的其他癌症类型，用于交叉预防或交叉治疗的其他癌症类型可为一种或多种。The whole cell components of the nano or micro vaccine system based on cancer cells and/or tumor tissue whole cell components of the present invention are prepared from one or more cancer cells or tumor tissues, and used for cross-prevention or cross-treatment is different from preparation The used cancer cells or other cancer types of tumor tissue can be one or more types of other cancers used for cross-prevention or cross-treatment.
本发明在将癌细胞和/或肿瘤组织裂解后首先获取在纯水或不含增溶剂的水溶液中可溶的水溶性组分，尔后采用含有增溶剂的增溶水溶液将水不溶性的组分溶解于增溶液中，这样就可将所有的细胞组分都转变为可在水溶液中溶解的组分并进而将其负载于纳米粒子或微米粒子内外以制备纳米疫苗或微米疫苗用于癌症的预防和治疗。在实际应用中也可将细胞或组织裂解后直接采用含有增溶剂的增溶水溶液溶解全细胞组分而不分别收集水溶性组分和非水溶性组分，并采用增溶水溶液溶解后的全细胞组分制备纳米疫苗或微米疫苗。In the present invention, after the cancer cells and/or tumor tissues are lysed, the water-soluble components that are soluble in pure water or an aqueous solution without a solubilizing agent are firstly obtained, and then the water-insoluble components are dissolved by a solubilizing aqueous solution containing a solubilizing agent In the solubilization solution, all the cell components can be converted into components that can be dissolved in the aqueous solution and then loaded inside and outside the nanoparticles or microparticles to prepare nano-vaccine or micro-vaccine for the prevention and treatment of cancer treat. In practical applications, cells or tissues can also be lysed directly with a solubilizing aqueous solution containing a solubilizing agent to dissolve the whole cell components without collecting the water-soluble components and water-insoluble components separately, and the whole cell components dissolved in the solubilizing aqueous solution can be used Cellular components make nanovaccine or microvaccine.
本发明通过采用含有增溶剂的水溶液将细胞中不溶于纯水或不含增溶剂水溶液的组分转化为在特定增溶溶液中可溶并可被用于制备纳米粒子和微米粒子，从而提高了纳米疫苗或微米疫苗所负载的抗原物质或成分的全面性和免疫原性。The present invention converts components insoluble in pure water or aqueous solutions without solubilizers in cells into soluble in specific solubilizing solutions and can be used to prepare nanoparticles and microparticles by using an aqueous solution containing a solubilizing agent, thereby improving The comprehensiveness and immunogenicity of the antigenic substances or components loaded by nano-vaccine or micro-vaccine.
本发明将癌细胞和/或肿瘤组织中全细胞组分分为可在纯水或不含增溶剂水溶液中溶解的水溶性部分和可用一定增溶剂溶解于水溶液中的非水溶性部分，并将水溶性部分和非水溶性部分包载于纳米粒子或微米粒子中和负载于其表面，从而保证了绝大部分抗原物质被负载于所制备的疫苗中。The present invention divides the whole cell components in cancer cells and/or tumor tissues into water-soluble parts that can be dissolved in pure water or aqueous solutions without solubilizers and water-insoluble parts that can be dissolved in aqueous solutions with certain solubilizers, and The water-soluble part and the water-insoluble part are entrapped in the nanoparticles or micro-particles and loaded on the surface thereof, thereby ensuring that most of the antigenic substances are loaded in the prepared vaccine.
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，也可细胞裂解前或（和）裂解后不经过任何灭活或（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或（和）变性处理，或者也可以细胞裂解前和裂解后均做灭活或（和）变性处理；本发明部分实施例中细胞裂解前或（和）裂解后的灭活或（和）变性处理方法为紫外照射和高温加热，在实际使用过程中也可以采用放射线辐照、高压、冷冻干燥和甲醛等灭活或变性处理方法。本领域技术人员可以理解，在实际应用过程中技术人员可根据具体情况进行适当调整。The whole cell components of the present invention can be inactivated or (and) denatured before or (and) after lysis to prepare nano-vaccine or micro-vaccine, or can not be processed before or (and) after lysis Any inactivation or (and) denaturation treatment can directly prepare nano-vaccine or micro-vaccine. In some embodiments of the present invention, the tumor tissue cells have been inactivated or (and) denatured before lysing. In actual use, they can also be inactivated or (and) denatured after cell lysis, or the cells can be lysed. Inactivation or (and) denaturation treatment before and after lysis; the inactivation or (and) denaturation treatment method before or (and) after lysis of cells in some embodiments of the present invention is ultraviolet irradiation and high temperature heating. Inactivation or denaturation treatment methods such as radiation irradiation, high pressure, freeze-drying and formaldehyde can also be used in the process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific conditions.
本发明所述全细胞组分的纳米疫苗或微米疫苗系统其结构示意图如图1-图19所示。在实际使用过程中可以为只使用其中的某一种特定结构的纳米粒子或微米粒子，或者是同时使用两种或两种以上的不同结构的纳米粒子或微米粒子。图1、图2中纳米粒子或微米粒子表面和内部均含有免疫增强佐剂；图3-图4中免疫增强剂只分布于纳米粒子或微米粒子的内部；图5-图6中纳米粒子或微米粒子只在外表面含有免疫增强佐剂；图7-图8纳米粒子或微米粒子内部和外表面均无免疫增强佐剂；图1A，图3A、图5A和图7A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；图1B，图3B，图5B和图7B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形成了一个内核部分，内核可为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2A，图4A，图6A和图8A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形成了多个内核部分，内核可为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2B，图4B，图6B和图8B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。The structural schematic diagrams of the nano-vaccine or micro-vaccine system of the whole cell component of the present invention are shown in Figures 1-19. In actual use, only nanoparticles or microparticles of a specific structure may be used, or two or more nanoparticles or microparticles of different structures may be used simultaneously. In Fig. 1 and Fig. 2, the surface and interior of nanoparticles or microparticles all contain immune enhancing adjuvants; in Fig. 3-Fig. Microparticles only contain immunoenhancing adjuvants on the outer surface; Figure 7-8 have no immune enhancing adjuvants on the inner and outer surfaces of nanoparticles or microparticles; Figure 1A, Figure 3A, Figure 5A and Figure 7A When the water-soluble or non-water-soluble components in the loaded cells or tissue components are distributed inside the nanoparticles or microparticles, no obvious inner core is formed; Figure 1B, Figure 3B, Figure 5B and Figure 7B show that the nanoparticles or microparticles The water-soluble or non-water-soluble components of the loaded cell or tissue components form a core part when they are distributed inside the nanoparticles or microparticles. The core can be generated during the preparation process or formed by using polymers or inorganic salts, etc. ; Fig. 2A, Fig. 4A, in Fig. 6A and Fig. 8A, the water-soluble component or the non-water-soluble component in the cell or tissue component loaded by nanoparticle or microparticle form multiple inner cores when they are distributed inside the nanoparticle or microparticle In part, the inner core can be generated during the preparation process or formed by using polymers or inorganic salts; Figure 2B, Figure 4B, Figure 6B and Figure 8B The water-soluble components of cells or tissue contained in nanoparticles or microparticles When the active component or water-insoluble component is distributed inside the nanoparticle or microparticle, it is located in the outer layer of the formed inner core; a: The inside and surface of the nanoparticle or microparticle are all water-soluble components in the cell or tissue components ; b: Nanoparticles or microparticles contain water-insoluble components in cells or tissue components; c: Nanoparticles or microparticles contain water-insoluble components in cells or tissue components The components loaded on the surface are all water-soluble components in the cell or tissue components; d: the water-soluble components contained in the nanoparticles or microparticles are the water-soluble components in the cells or tissue components, and all the components loaded on the surface are cells or tissues The water-insoluble components in the components; e: the water-soluble components and water-insoluble components in the cells or tissue components that are simultaneously contained in the nanoparticles or microparticles, and the cells or tissues are also loaded on the surface of the nanoparticles or microparticles The water-soluble components and water-insoluble components in the components; f: the water-soluble components and water-insoluble components in the cells or tissue components that are simultaneously contained inside the nanoparticles or microparticles, while the surface of the nanoparticles or microparticles is only loaded Water-soluble components in cells or tissue components; g: water-soluble components and water-insoluble components in cells or tissue components contained inside nanoparticles or microparticles, while the surface of nanoparticles or microparticles only supports cells or water-insoluble components in tissue components; h: only the water-insoluble components in cells or tissue components contained inside nanoparticles or microparticles, while the surface of nanoparticles or microparticles simultaneously loads the water-insoluble components in cells or tissue components Water-soluble components and water-insoluble components; i: the water-soluble components in the cells or tissue components contained only in the interior of nanoparticles or microparticles, while the surface of nanoparticles or microparticles is the same Water-soluble components and water-insoluble components in cell or tissue components are loaded at the same time.
图9-图10中纳米粒子或微米粒子表面和内部均含有免疫佐剂；图11-图12中免疫佐剂只分布于纳米粒子或微米粒子的内部；图13-图14中纳米粒子或微米粒子只在外表面含有免疫佐剂；图15-图16纳米粒子或微米粒子内部和外表面均无免疫佐剂；图17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18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19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9-16中，图9中2.a-2.i，图11中6.a-6.i，图13中10.a-10.i和图15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10中3.a-3.i，图11中7.a-7.i，图13中11.a-11.i和图15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10中4.a-4.i，图12中8.a-8.i，图14中12.a-12.i和图16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10中5.a-5.i，图12中9.a-9.i，图14中13.a-13.i和图16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17-19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。In Figure 9-Figure 10, the surface and interior of nanoparticles or microparticles contain immune adjuvants; in Figure 11-Figure 12, immune adjuvants are only distributed in the interior of nanoparticles or microparticles; Particles only contain immune adjuvant on the outer surface; Figure 15-Figure 16 has no immune adjuvant inside and outside the nanoparticle or microparticle; Figure 17 cell components and/or immune adjuvant are only distributed inside the nanoparticle or microparticle; Figure 18 Cell components and/or immune adjuvants are only distributed outside the nanoparticles or microparticles; Figure 19 Cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively. In Figures 9-16, 2.a-2.i in Figure 9, 6.a-6.i in Figure 11, 10.a-10.i in Figure 13 and 14.a-14.i in Figure 15 When the water-soluble or insoluble components in the cells or tissue components loaded by nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, no obvious inner core is formed; 3.a-3.i in Fig. 10, Fig. 7.a-7.i in 11, 11.a-11.i in Figure 13 and 15.a-15.i in Figure 15. Water-soluble components in the cells or tissue components loaded by nanoparticles or microparticles Or the water-insoluble component is distributed in a core part inside the nanoparticle or microparticle; 4.a-4.i in Figure 10, 8.a-8.i in Figure 12, 12.a-12.i in Figure 14 And in Fig. 16 16.a-16.i the water-soluble component or the non-water-soluble component in the cell or the tissue component that nanoparticle or microparticle are loaded are distributed in a plurality of inner core parts of nanoparticle or microparticle interior; Fig. 10 In 5.a-5.i, 9.a-9.i in Figure 12, 13.a-13.i in Figure 14 and 17.a-17.i in Figure 16 contained in nanoparticles or microparticles The water-soluble or non-water-soluble components in the cells or tissue components are distributed in the outer layer of the inner core formed inside the nanoparticles or micro-particles; a: both the internal and surface loading of nanoparticles or micro-particles are cells or tissue groups the water-soluble components in the component; b: the nanoparticle or microparticle internally and surface-loaded are all water-insoluble components in the cell or tissue components; c: the nanoparticle or microparticle internally encapsulated is the cell or tissue The water-insoluble components in the components are all water-soluble components in the cell or tissue components; d: the water-soluble components in the cells or tissue components are loaded on the surface of the nanoparticles or microparticles are the water-insoluble components in the cell or tissue components; e: the water-soluble components and the water-insoluble components in the cells or tissue components contained inside the nanoparticles or microparticles, while the surface of the nanoparticles or microparticles It also loads water-soluble components and water-insoluble components in cells or tissue components at the same time; f: water-soluble components and water-insoluble components in cells or tissue components that are simultaneously contained in nanoparticles or microparticles, while nanoparticles or the surface of microparticles is only loaded with water-soluble components in cells or tissue components; g: the water-soluble components and water-insoluble components in cells or tissue components contained in nanoparticles or microparticles at the same time, while nanoparticles or microparticles The surface of the particle is only loaded with water-insoluble components in cells or tissue components; h: the interior of nanoparticles or microparticles is only loaded with insoluble components in cells or tissue components, while the surface of nanoparticles or microparticles is loaded with cells at the same time or water-soluble components and water-insoluble components in tissue components; i: the inside of nanoparticles or microparticles only contains water-soluble components in cells or tissue components, while the surface of nanoparticles or microparticles simultaneously loads cells or tissues Water-soluble and water-insoluble ingredients in the components. In Figures 17-19, the water-soluble or water-insoluble components of the cells or tissue components loaded by nanoparticles or microparticles in a, b and c do not form an obvious inner core when they are distributed inside the nanoparticles or microparticles ; The water-soluble components or water-insoluble components in the cells or tissue components loaded by nanoparticles or microparticles in d, e and f are distributed in a core part inside the nanoparticles or microparticles; g, h and i The water-soluble components or water-insoluble components in the cells or tissue components loaded by particles or microparticles are distributed in multiple core parts inside nanoparticles or microparticles; j, k and l are contained in nanoparticles or microparticles The water-soluble or non-water-soluble components in the cell or tissue components are distributed in the outer layer of the inner core formed by nanoparticles or microparticles; a, d, The nanoparticles or microparticles in g and j are loaded with water-soluble components in cells or tissue components; the nanoparticles or microparticles in b, e, h and k are loaded with water-insoluble components in cells or tissue components. properties; c, f, i, and l, nanoparticles or microparticles simultaneously load water-soluble components and water-insoluble components in cells or tissue components.
在本发明所述制备基于全细胞组分的疫苗系统的方法为常用制备方法。在一些实施方案中，制备纳米或微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为有机高分子聚乳酸-羟基乙酸共聚物（PLGA）, 所采用的免疫佐剂为 poly(I:C)、卡介苗（BCG）、锰佐剂或CpG。本领域技术人员可以理解，在实际应用过程中技术人员可根据具体情况对制备方法、制备过程、所采用的纳米粒子或微米粒子的制备材料、免疫佐剂的种类和浓度等进行适当调整。The method for preparing the vaccine system based on whole cell components described in the present invention is a common preparation method. In some embodiments, the double emulsion method in the solvent evaporation method is used to prepare nano or micro vaccines. The nanoparticle preparation material used is organic polymer polylactic acid-glycolic acid copolymer (PLGA), and the immune adjuvant used is poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant, or CpG. Those skilled in the art can understand that in the actual application process, the skilled person can make appropriate adjustments to the preparation method, preparation process, nanoparticle or microparticle preparation material used, type and concentration of immune adjuvant, etc. according to specific conditions.
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高分子材料的有机相中。In some embodiments, the specific preparation method of the double-emulsion method used in the present invention is as follows: Step 1, adding a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of a medical solution containing a second predetermined concentration. In the organic phase of polymer materials.
在一些实施例中，水相溶液可含有癌细胞和/或肿瘤组织裂解物中的各组分以及免疫增强佐剂poly(I:C)、BCG、锰佐剂或CpG；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增溶剂中的原非水溶性组分；或者是溶解于增溶剂中的全细胞组分。水相溶液所含有来自癌细胞和/或肿瘤组织的水溶性组分的浓度或者是来自癌细胞和/或肿瘤组织的溶于增溶剂中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1 ng/mL，能负载足够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0.01 ng/mL。In some embodiments, the aqueous phase solution may contain the components in the cancer cell and/or tumor tissue lysate and the immunoenhancing adjuvant poly(I:C), BCG, manganese adjuvant or CpG; each component in the lysate The components are respectively water-soluble components or original water-insoluble components dissolved in a solubilizing agent; or whole cell components dissolved in a solubilizing agent. The concentration of the water-soluble components from cancer cells and/or tumor tissues contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in the solubilizer from cancer cells and/or tumor tissues, that is, the first The predetermined concentration requires that the concentration of protein and peptide is greater than 1 ng/mL, enough antigen can be loaded to activate the relevant immune response. The concentration of the immune enhancing adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
在一些实施例中，水相溶液含有癌细胞和/或肿瘤组织裂解物中的各组分以及免疫增强佐剂poly(I:C)，BCG、锰佐剂或CpG；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增溶剂中的原非水溶性组分。水相溶液所含有的水溶性组分的浓度或者是来自溶于增溶剂中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1 ng/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0.01 ng/mL。In some embodiments, the aqueous phase solution contains each component in the cancer cell and/or tumor tissue lysate and the immunoenhancing adjuvant poly(I:C), BCG, manganese adjuvant or CpG; each group in the lysate The components are respectively water-soluble components or original water-insoluble components dissolved in the solubilizing agent during preparation. The concentration of the water-soluble components contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in the solubilizer, that is, the first predetermined concentration requires that the concentration of the protein polypeptide be greater than 1 ng/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 0.01 ng/mL.
在本发明中，将医用高分子材料溶解于有机溶剂中，得到第二预定体积的含有第二预定浓度医用高分子材料的有机相。在一些实施例中，医用高分子材料为PLGA，有机溶剂选用二氯甲烷。另外，在一些实施例中，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100 mg/mL。In the present invention, the medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing a second predetermined concentration of the medical polymer material. In some embodiments, the medical polymer material is PLGA, and the organic solvent is dichloromethane. In addition, in some embodiments, the range of the second predetermined concentration of the medical polymer material is 0.5mg/mL-5000mg/mL , preferably 100 mg/mL.
在本发明中，之所以选择PLGA或修饰的PLGA，是由于该材料为生物可降解材料且已被FDA批准用作药物辅料。研究表明PLGA具有一定的免疫调节功能，因而适合作为疫苗制备时的辅料。In the present invention, PLGA or modified PLGA is selected because the material is a biodegradable material and has been approved by the FDA as a drug excipient. Studies have shown that PLGA has a certain immune regulation function, so it is suitable as an auxiliary material for vaccine preparation.
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。In practice, the second predetermined volume of the organic phase is set according to its ratio with the first predetermined volume of the aqueous phase. In the present invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges 1:1.1-1:5000, preferably 1:10. During the specific implementation process, 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 required to adjust the size of the prepared nanoparticles or microparticles.
步骤2，将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或进行均质化处理或者采用微流控处理。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50 rpm～500 rpm，搅拌时间大于1分钟，比如60～600秒。In step 2, the mixed solution obtained in step 1 is subjected to ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or homogenization treatment or microfluidic treatment. This step is for nanometerization or micronization. The length of ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles. Too long or too short will bring about changes in particle size. Therefore, it is necessary to select an appropriate ultrasonic time. . In the present invention, the ultrasonic time is greater than 0.1 second, such as 2-200 seconds, the stirring speed is greater than 50 rpm, such as 50 rpm-500 rpm, and the stirring time is greater than 1 minute, such as 60-600 seconds.
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化处理或者采用微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌或均质化处理或者微流控处理进行纳米化或微米化。Step 3, adding the mixture obtained after the treatment in step 2 into a third predetermined volume of an aqueous solution containing an emulsifier of a third predetermined concentration and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or using micro Flow control processing. In this step, the mixture obtained in step 2 is added to the emulsifier aqueous solution to continue ultrasonication or stirring or homogenization treatment or microfluidic treatment for nanometerization or micronization.
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5 mL，第三预定浓度为20 mg/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，优先地可以为2:5。在具体实施过程中为了控制纳米或微米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。In the present invention, the emulsifier aqueous solution is polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the range between the second predetermined volume and the third predetermined volume is 1:1.1 -1:1000 for setting, preferably 2:5. In order to control the size of the nano or micro particles during the specific implementation process, the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
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。Similarly, the ultrasonic time or stirring time in this step, the volume and concentration of the emulsifier aqueous solution are all based on the purpose of obtaining nanoparticles or microparticles of appropriate size.
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，并进行搅拌和/或真空处理直至满足预定条件。Step 4, adding the liquid obtained after the treatment in step 3 into a fourth predetermined volume of an emulsifier aqueous solution of a fourth predetermined concentration, and performing stirring and/or vacuum treatment until predetermined conditions are met.
本步骤中，乳化剂水溶液依然为PVA， 第四预定浓度为5 mg/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。In this step, the emulsifier aqueous solution is still PVA, and the fourth predetermined concentration is 5 mg/mL, the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of appropriate size. The selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In order to control the size of nanoparticles or microparticles during specific implementation, the ratio between the third predetermined volume and the fourth predetermined volume can be adjusted.
在本发明中，本步骤的预定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。In the present invention, the predetermined condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.
步骤5，将步骤4处理满足预定条件的混合液在以大于100 RPM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。Step 5, after centrifuging the mixed solution that meets the predetermined conditions in step 4 at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in the fifth predetermined volume of the first Five predetermined concentrations of the aqueous solution containing the lyoprotectant or the sixth predetermined volume of PBS (or physiological saline).
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和/或肿瘤组织裂解物的相关实验。In some embodiments of the present invention, when the precipitate obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline), freeze-drying is not required, and subsequent nanoparticle or microparticle surface adsorption of cancer cells and/or Related experiments on tumor tissue lysates.
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和/或肿瘤组织裂解物的相关实验。In some embodiments of the present invention, when the precipitate obtained in step 5 is resuspended in an aqueous solution containing a lyoprotectant, it needs to be lyophilized, and after lyophilization, the subsequent adsorption of cancer cells and/or tumors on the surface of nanoparticles or microparticles Related experiments on tissue lysates.
在本发明中，所述冻干保护剂选用海藻糖（Trehalose）。In the present invention, the lyoprotectant is selected from trehalose (Trehalose).
在本发明中，该步骤的冻干保护剂的第五预定体积为20 mL，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。In the present invention, the fifth predetermined volume of the lyoprotectant in this step is 20 mL, and the fifth predetermined concentration is 4% by mass. The reason for this setting is to not affect the effect of lyophilization in subsequent lyophilization. .
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。In step 6, the suspension containing the lyoprotectant obtained in step 5 is lyophilized, and the lyophilized substance is used for future use.
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/微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/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尿素中的原非水溶性组分混合后即得纳米疫苗或微米疫苗，为基于全细胞组分的疫苗系统。Step 7, resuspend the sixth predetermined volume of the nanoparticle/microparticle-containing suspension obtained in step 5 in PBS (or normal saline) or resuspend with the sixth predetermined volume of PBS (or normal saline) The freeze-dried freeze-dried substance containing nanoparticles/microparticles and freeze-drying protective agent obtained in step 6 is mixed with the seventh predetermined volume of water-soluble components or the original water-insoluble components dissolved in 8M urea. Nano vaccines or micro vaccines are vaccine systems based on whole cell components.
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。In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferential volume ratio is 1:100 to 100:1, and the optimum volume ratio is 1:30 to 30:1 .
在一些实施例中，所述重悬的纳米粒子或微米粒子混悬液体积为10 mL，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8M尿素中的原非水溶性组分的体积与为1 mL。二者所需体积和比例可在应用中调节。In some embodiments, the resuspended nanoparticle or microparticle suspension has a volume of 10 mL, contains cancer cell lysate or contains water-soluble components in tumor tissue lysate or original non-alcohol dissolved in 8M urea. The volume of the water-soluble component is 1 mL. The required volume and ratio of the two can be adjusted in the application.
在本发明中，所采用的含有癌细胞和/或肿瘤组织裂解物中水溶性组分或者溶于8M尿素中的原非水溶性组分中含有poly(I:C)、卡介苗（BCG）、锰佐剂或CpG，且poly(I:C)、BCG或CpG的浓度为大于1 ng/mL。In the present invention, the water-soluble components in the cancer cell and/or tumor tissue lysates or the original non-water-soluble components dissolved in 8M urea contain poly(I:C), Bacillus Calmette-Guerin (BCG), Manganese adjuvant or CpG, and the concentration of poly(I:C), BCG or CpG is greater than 1 ng/mL.
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和激活免疫反应，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。The particle size of nano-vaccine or micro-vaccine is nano-scale or micron-scale, which can ensure that the vaccine is phagocytized by antigen-presenting cells and activates the immune response. In order to improve the phagocytosis efficiency, the particle size should 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 1μm-1000μm, more preferably, The particle size is 1 μm-100 μm, more preferably, the particle size is 1 μm-10 μm, most preferably, the particle size is 1 μm-5 μm. In this embodiment, the particle size of the nanoparticle vaccine is 100nm-600nm, and the particle size of the micron vaccine is 1 μm-5 μm.
另外，在本发明中，采用尿素和盐酸胍来增溶癌细胞和/或肿瘤组织裂解物中的原非水溶性组分，在实际使用中亦可使用任何其他可使癌细胞和/或肿瘤组织裂解物中的原非水溶性组分溶解于水溶液的增溶物质，如脱氧胆酸钠，SDS，pH大于7的碱性溶液，pH小于7的酸性溶液，白蛋白，卵磷脂、高浓度无机盐、Triton、吐温、DMSO、乙腈、乙醇、甲醇、DMF、异丙醇、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、IGEPAL®CA-630；或者也可以使用上述增溶液同时溶解水溶性组分和非水溶性组分。 In addition, in the present invention, urea and guanidine hydrochloride are used to solubilize the original water-insoluble components in the cancer cell and/or tumor tissue lysate, and any other components that can make the cancer cell and/or tumor The original water-insoluble components in the tissue lysate are dissolved in the solubilizing substances of the aqueous solution, such as sodium deoxycholate, SDS, alkaline solution with pH greater than 7, acidic solution with pH less than 7, albumin, lecithin, high concentration Inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630; or the above-mentioned solubilizing solution can be used to dissolve both water-soluble components and water-insoluble components.
另外，在本发明中，采用8M的尿素和6M的盐酸胍水溶液来增溶癌细胞和/或肿瘤组织裂解物中的原非水溶性组分，在实际使用中亦可使用任何其他可使癌细胞和/或肿瘤组织裂解物中的原非水溶性组分溶解于水溶液的尿素浓度或盐酸胍浓度；或者使用8M尿素水溶液或6M的盐酸胍水溶液等增溶剂同时溶解水溶性组分和非水溶性组分。In addition, in the present invention, 8M urea and 6M guanidine hydrochloride aqueous solution are used to solubilize the original water-insoluble components in cancer cells and/or tumor tissue lysates, and any other components that can make cancer cells The original water-insoluble components in cell and/or tumor tissue lysates are dissolved in the urea concentration or guanidine hydrochloride concentration of the aqueous solution; sex component.
另外，在本发明实施例中，纳米疫苗和微米疫苗的制备采用复乳法，在实际中也可采用任何其他常用的纳米粒子或微米粒子制备方法。In addition, in the embodiment of the present invention, the preparation of nano-vaccine and micro-vaccine adopts the double emulsion method, but any other commonly used method for preparing nanoparticles or micro-particles can also be used in practice.
另外，在本发明实施例中，纳米疫苗和微米疫苗的制备材料为PLGA，在实际中亦可采用任何其他可以制备纳米粒子或微米粒子的材料。In addition, in the embodiment of the present invention, the preparation material of the nano-vaccine and the micro-vaccine is PLGA, but any other material capable of preparing nanoparticles or micro-particles can also be used in practice.
另外，在本发明实施例中，癌细胞和/或肿瘤组织裂解物中水溶性组分或者溶于8M尿素中的原非水溶性组分分别包载在纳米/微米粒子内部和吸附在纳米/微米粒子表面，在实际使用时，癌细胞和/或肿瘤组织裂解物中水溶性组分和溶于8M尿素中的原非水溶性组分亦可混合后再包载到粒子内部或吸附到粒子表面；或者也可以采用8M尿素同时溶解水溶性组分和非水溶性组分然后包载于纳米粒子或微米粒子内部和/或吸附于纳米粒子或微米粒子表面。In addition, in the embodiment of the present invention, the water-soluble components in the cancer cell and/or tumor tissue lysates or the original water-insoluble components dissolved in 8M urea are respectively entrapped inside the nano/micro particles and adsorbed on the nano/micro particles. On the surface of micron particles, in actual use, the water-soluble components in cancer cell and/or tumor tissue lysates and the original water-insoluble components dissolved in 8M urea can also be mixed and then packed into the particles or adsorbed to the particles surface; or 8M urea can also be used to simultaneously dissolve the water-soluble component and the water-insoluble component and then entrapped inside the nanoparticle or microparticle and/or adsorbed on the surface of the nanoparticle or microparticle.
另外，在本发明实施例中，采用poly(I:C)、卡介苗（BCG）、锰佐剂和CpG为免疫佐剂，在实际中亦可不加入免疫佐剂或者加入任何其他具有免疫增强功能的免疫佐剂，如模式识别受体激动剂、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、植物油、内毒素、脂质体佐剂、GM-CSF、MF59、双链RNA、双链DNA、铝佐剂、CAF01、人参、黄芪等中药有效成分。In addition, in the embodiment of the present invention, poly(I:C), Bacillus Calmette-Guerin (BCG), manganese adjuvant and CpG are used as immune adjuvants. Immune adjuvants, such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanolic extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyantibody A, mineral oil, virus-like particles, reconstitution for immune enhancement Influenza virion, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant poly ICLC, corynebacterium brevis vaccine, hemolytic chain Bacillus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, double-stranded RNA, double-stranded DNA, aluminum adjuvant, CAF01, ginseng, astragalus and other active ingredients of traditional Chinese medicine.
另外，在本发明中，部分实施例中采用的疫苗为纳米疫苗，部分实施例采用的是微米疫苗。本领域技术人员在实际中可以根据实际情况选择采用纳米疫苗或微米疫苗，即基于癌细胞和/或肿瘤组织全细胞组分的疫苗系统。In addition, in the present invention, the vaccines used in some embodiments are nano vaccines, and some embodiments use micro vaccines. Those skilled in the art can choose to use nano-vaccine or micro-vaccine according to the actual situation, that is, a vaccine system based on whole cell components of cancer cells and/or tumor tissues.
本发明基于全细胞组分的疫苗系统由全细胞组分、纳米/微米粒子组成，或者由全细胞组分、纳米/微米粒子、免疫增强佐剂组成。为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The vaccine system based on whole cell component of the present invention is composed of whole cell component, nano/micro particle, or composed of whole cell component, nano/micro particle and immune enhancing adjuvant. In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. . Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
如无特殊说明，本发明实施例中所使用的方法均为常规方法；所使用的材料、试剂等均可从商业途径得到。本发明实施例中所涉及到的纳米粒子或微米粒子结构、制备方法、疾病治疗时的使用策略等仅为代表性方法，其他纳米粒子或微米粒子结构、制备方法、疾病预防或治疗时的使用策略、与其他药物的联用策略亦可采用本发明所述的方法。实施例中仅列出了本发明在部分癌症中的应用，但是本发明亦可用在其他类型的任何癌症。对于实施例中所用到的具体方法或材料，本领域技术人员可以在本发明技术思路的基础上，根据已有的技术进行常规的替换选择，而不仅限于本发明实施例的具体记载。在实际应用时具体给药时间、给药次数、给药方案、与其他药物联用情况可根据情况调整。Unless otherwise specified, the methods used in the examples of the present invention are conventional methods; the materials and reagents used can be obtained from commercial sources. The nanoparticle or microparticle structure, preparation method, and use strategy for disease treatment involved in the embodiments of the present invention are only representative methods, and the use of other nanoparticle or microparticle structures, preparation methods, and disease prevention or treatment The strategy and the combination strategy with other drugs can also adopt the method described in the present invention. The examples only list the application of the present invention in some cancers, but the present invention can also be used in any other types of cancer. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional replacements based on the technical ideas of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention. In actual application, the specific administration time, administration frequency, administration regimen, and combination with other drugs can be adjusted according to the actual situation.
实施例1 肺癌肿瘤组织全细胞组分负载于纳米粒子内部和表面用于黑色素瘤的预防：本实施例以B16F10小鼠黑色素瘤为癌症模型来说明如何制备负载有肺癌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。首先裂解LLC肺癌组织瘤块制备肺癌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Polyinosinic-polycytidylic acid (poly(I:C))为免疫佐剂采用溶剂挥发法制备负载有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。Example 1 The whole cell components of lung cancer tumor tissue are loaded inside and on the surface of nanoparticles for the prevention of melanoma: this example uses B16F10 mouse melanoma as a cancer model to illustrate how to prepare the whole cell components of lung cancer tumor tissue Nano-vaccine, and apply the vaccine to prevent melanoma. First, the tumor block of LLC lung cancer tissue is cracked to prepare water-soluble components and water-insoluble components of lung cancer tumor tissue. Then, the organic polymer material PLGA was used as the nanoparticle framework material, and Polyinosinic-polycytidylic acid (poly(I:C)) was used as the immune adjuvant to prepare nanoparticles loaded with water-soluble components and water-insoluble components by solvent evaporation method. vaccine. The nanovaccine was then employed to prevent melanoma.
（1）肺癌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2×10 6个LLC肺癌细胞，在肿瘤长到体积分别为约1000 mm 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纯水并反复冻融5次，并超声裂解细胞。待细胞裂解后，将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分；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。上述所得来源于肺癌肿瘤组织裂解物的水溶性组分和溶解于8M尿素中的原非水溶性组分即为肺癌肿瘤组织制备的用于预防黑色素瘤的纳米疫苗的原料来源。 (1) Lysis of lung cancer tissue and collection of various components: 2× 106 LLC lung cancer cells were subcutaneously inoculated on the back of each C57BL/6 mouse, and the mice were sacrificed when the tumors grew to a volume of about 1000 mm3 , respectively. Remove tumor tissue. After the tumor tissue was cut into pieces and ground, pure water was added through a cell strainer and repeated freezing and thawing 5 times, and the cells were lysed by ultrasound. After the cells are lysed, centrifuge the lysate at a speed of 5000g for 5 minutes and take the supernatant, which is the water-soluble component soluble in pure water; The water-insoluble components of water were converted to be soluble in 8M aqueous urea solution. The above-mentioned water-soluble components derived from lung cancer tumor tissue lysate and the original water-insoluble components dissolved in 8M urea are the raw materials for the nano-vaccine prepared from lung cancer tumor tissue for preventing melanoma.
将2×10 6个LLC肺癌细胞更换为1.5×10 5个B16F10细胞，再如上同样的方法，制备来源于黑色素瘤肿瘤组织裂解物的水溶性组分和溶解于8M尿素中的原非水溶性组分，为黑色素瘤肿瘤组织制备的用于预防黑色素瘤的纳米疫苗的原料来源。 Replace 2× 106 LLC lung cancer cells with 1.5× 105 B16F10 cells, and prepare the water-soluble components derived from melanoma tumor tissue lysates and the original water-insoluble components dissolved in 8M urea in the same way as above The component is the raw material source of the nano vaccine used to prevent melanoma prepared from melanoma tumor tissue.
（2）纳米疫苗的制备：本实施例中制备纳米疫苗及作为对照的空白纳米粒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，所采用的免疫佐剂为poly(I:C) 且poly(I:C)既分布于纳米粒子内部也吸附于纳米粒子表面。制备方法如前所述。在纳米粒子表面负载细胞组分和免疫佐剂之前纳米粒子平均粒径为320nm，纳米粒子表面吸附细胞组分和免疫佐剂后所得纳米疫苗平均粒径为340nm，纳米疫苗表面电位为-5mV左右。每1mg PLGA纳米粒子负载180μg蛋白质或多肽组分，每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂共约为0.01mg且内外各半。空白纳米粒粒径为290nm，空白纳米粒制备时分别采用含有等量poly（I:C）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，空白纳米粒子外表面吸附与纳米疫苗等量的poly(I:C)。(2) Preparation of nano-vaccine: In this example, the preparation of nano-vaccine and the blank nano-particles used as a control adopt the double emulsion method in the solvent evaporation method, and the molecular weight of the nano-particle preparation material PLGA used is 24KDa-38KDa. The adjuvant is poly(I:C) And poly(I:C) is not only distributed inside the nanoparticles but also adsorbed on the surface of the nanoparticles. The preparation method is as described above. Before loading cell components and immune adjuvant on the surface of nanoparticles, the average particle size of nanoparticles is 320nm, and the average particle size of nano-vaccine after adsorption of cell components and immune adjuvant on the surface of nanoparticles is 340nm, and the surface potential of nano-vaccine is about -5mV . Each 1mg PLGA nanoparticle is loaded with 180μg protein or polypeptide component, and the poly(I:C) immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.01mg in total, and the inside and outside are divided in half. The particle size of blank nanoparticles is 290nm. When preparing blank nanoparticles, pure water or 8M urea containing the same amount of poly (I:C) is used to replace the corresponding water-soluble components and non-water-soluble components. The outer surface of blank nanoparticles Adsorb the same amount of poly(I:C) as the nano-vaccine.
（3）纳米疫苗用于癌症的治疗：本研究对照组分别是PBS组、空白纳米粒+肿瘤组织裂解物组。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。(3) Nano-vaccine for the treatment of cancer: The control groups in this study were PBS group, blank nanoparticles + tumor tissue lysate group. Select 6-8-week-old female C57BL/6 as model mice to prepare melanoma-bearing mice.
使用肺癌肿瘤组织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内部和表面都负载肺癌肿瘤组织裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子；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。 The dosage regimen of the lung cancer tumor tissue nano-vaccine group is as follows: 200 μL of lung cancer tumor tissue lysates were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. 2 mg PLGA nanoparticles of water-soluble components and 200 μL of 2 mg PLGA nanoparticles loaded with original water-insoluble components dissolved in 8M urea in 200 μL; on day 0, 1.5×10 5 were subcutaneously inoculated in the lower right lower back of each mouse B16F10 cells.
使用黑色素瘤肿瘤组织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内部和表面都负载黑色素瘤肿瘤组织裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子；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。 The dosing regimen of the melanoma tumor tissue nanovaccine group is as follows: 200 μL of melanoma tumor tissue was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. 2 mg PLGA nanoparticles of the water-soluble component in the lysate and 200 μL of 2 mg PLGA nanoparticles of the original water-insoluble component dissolved in 8 M urea were loaded on the inside and on the surface; on day 0, 1.5 × 10 5 B16F10 cells.
PBS空白对照组方案如下：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400μL 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。 The protocol of the PBS blank control group was as follows: 400 μL of PBS was injected subcutaneously on the 49th day, 42th day, 35th day, 28th day and 14th day before melanoma inoculation. On day 0, 1.5× 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
空白纳米粒+组织裂解物对照组：在接种黑色素瘤之前49天、42天、35天、28天和14天分别皮下注射400μL 空白纳米粒和游离裂解物（与纳米疫苗组等量）；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。 Blank nanoparticles + tissue lysate control group: 400 μL of blank nanoparticles and free lysate (equal to nanovaccine group) were subcutaneously injected 49 days, 42 days, 35 days, 28 days and 14 days before melanoma inoculation; Nanoparticles and free lysates were injected at different sites; on day 0, 1.5 × 105 B16F10 cells were subcutaneously inoculated on the lower right back of each mouse.
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。 In the experiment, the size of the mouse tumor volume was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图20所示，肺癌肿瘤组织纳米疫苗处理组小鼠的肿瘤在接种后消失，而PBS对照组和空白纳米粒对照组小鼠肿瘤都长大。与PBS空白对照组，空白纳米粒+细胞裂解物对照组相比，疫苗处理组（Vaccines）小鼠在肿瘤生长速度和小鼠生存期都有显著性差异。肺癌肿瘤组织制备的纳米疫苗对黑色素瘤的预防效果好于黑色素瘤肿瘤组织制备的纳米疫苗。综上所述，本发明所述的负载肺癌肿瘤组织的水溶性组分和非水溶性组分的纳米疫苗对黑色素瘤具有交叉预防效果。(4) Experimental results: As shown in Figure 20, the tumors of the mice in the lung cancer tumor tissue nano-vaccine treatment group disappeared after inoculation, while the tumors in the mice in the PBS control group and the blank nanoparticle control group grew up. Compared with the PBS blank control group and the blank nanoparticle + cell lysate control group, the vaccine treatment group (Vaccines) mice had significant differences in tumor growth rate and mouse survival period. The nano-vaccine prepared from lung cancer tumor tissue has a better preventive effect on melanoma than the nano-vaccine prepared from melanoma tumor tissue. In summary, the nano-vaccine loaded with water-soluble components and water-insoluble components of lung cancer tumor tissue according to the present invention has a cross-preventive effect on melanoma.
实施例2 肺癌细胞水溶性组分负载于微米粒子内部和表面用于黑色素瘤预防：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LLC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。Example 2 The water-soluble components of 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 microparticles loaded with only the water-soluble components of LLC lung cancer cell components Micron vaccine, and the use of the vaccine to prevent melanoma.
本实施例中，首先裂解LLC肺癌细胞以制备LLC肺癌细胞的水溶性组分和非水溶性组分。然后以高分子材料为微米粒子骨架材料，以CpG为免疫佐剂制备负载有LLC细胞水溶性组分的微米疫苗。并采用该疫苗预防黑色素瘤。In this example, LLC lung cancer cells were firstly lysed to prepare water-soluble components and water-insoluble components of LLC lung cancer cells. Then, the micron vaccine loaded with the water-soluble components of LLC cells is prepared by using the polymer material as the micron particle skeleton material and CpG as the immune adjuvant. And use the vaccine to prevent melanoma.
（1）癌细胞的裂解及各组分的收集：收集LLC肺癌细胞，去除培养基后采用-20℃冷冻，加超纯水后反复冻融3次，并超声裂解细胞。待细胞裂解后，将裂解物以3000g的转速离心5min取上清液即为LLC肺癌细胞中可溶于纯水的水溶性组分。上述所得来源于肺癌细胞裂解物的水溶性组分即为肺癌细胞制备的微米疫苗的原料来源。(1) Lysis of cancer cells and collection of components: Collect LLC lung cancer cells, remove the culture medium, freeze at -20°C, add ultrapure water, freeze and thaw three times, and lyse the cells by ultrasonic. After the cells were lysed, the lysate was centrifuged at a speed of 3000g for 5 minutes to obtain the supernatant, which was the water-soluble fraction in the LLC lung cancer cells that could be dissolved in pure water. The water-soluble component derived from the lung cancer cell lysate obtained above is the raw material source of the micron vaccine prepared by the lung cancer cell.
将LLC肺癌细胞更换为B16F10，如上同样的方法，制备来源于黑色素瘤细胞裂解物的水溶性组分，为黑色素瘤细胞制备的微米疫苗的原料来源。The LLC lung cancer cells were replaced with B16F10, and the water-soluble fraction derived from the melanoma cell lysate was prepared by the same method as above, which was the raw material source of the micron vaccine prepared by the melanoma cells.
（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有机高分子材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为 CpG 且CpG既分布于微米粒子内部也吸附于微米粒子表面。制备方法如前所述。在微米粒子表面吸附细胞组分和免疫佐剂后所得微米疫苗粒径为1.30 μm左右，微米粒子平均表面电位为-5mV左右。每1 mg PLGA微米粒子负载200μg蛋白质或多肽组分，每1mgPLGA微米粒内外所使用的CpG免疫佐剂为0.01mg且内外各半。空白微米粒粒径为1.25 μm左右，空白微米粒制备时分别采用含有等量CpG的纯水代替相对应的水溶性组分。(2) Preparation of micron vaccines: In this example, the preparation of micron vaccines and the blank micron particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micron particle preparation material used is an organic polymer material PLGA with a molecular weight of 38KDa-54KDa , the immune adjuvant used is CpG, and CpG is not only distributed inside the microparticles but also adsorbed on the surface of the microparticles. The preparation method is as described above. The micron vaccine particle size obtained after adsorbing cell components and immune adjuvant on the surface of the micron particle is about 1.30 μm, and the average surface potential of the micron particle is about -5mV. Each 1 mg of PLGA microparticles is loaded with 200 μg of protein or polypeptide components, and the CpG immune adjuvant used inside and outside of each 1 mg of PLGA microparticles is 0.01 mg, and the inside and outside are divided into half. The particle size of the blank microparticles is about 1.25 μm, and the corresponding water-soluble components are replaced by pure water containing the same amount of CpG when the blank microparticles are prepared.
（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。(3) Micron vaccine for cancer prevention: select 6-8 week old female C57BL/6 to prepare melanoma tumor-bearing mice.
微米疫苗组方案如下：在接种黑色素瘤之前第28天、第21天、第14天分别皮下注射400μL内部和表面都负载癌细胞裂解物中水溶性成分的4mg PLGA微米粒子。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。 The micro-vaccine group scheme is as follows: 400 μL of 4 mg PLGA micro-particles loaded with water-soluble components in cancer cell lysates were subcutaneously injected on the 28th, 21st, and 14th days before inoculation of melanoma, respectively. On day 0, 1.5× 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
PBS空白对照组方案如下：在接种黑色素瘤之前第28天、21天、14天分别皮下注射400 μL 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。 The protocol of the PBS blank control group was as follows: 400 μL of PBS were subcutaneously injected on the 28th, 21st, and 14th days before melanoma inoculation. On day 0, 1.5× 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
空白微米粒+细胞裂解物对照组：在接种黑色素瘤之前第28天、第21天、第14天分别皮下注射400μL空白微米粒子和与疫苗中等量的癌细胞裂解物。Blank microparticles+cell lysate control group: 400 μL of blank microparticles and cancer cell lysate equal to that in the vaccine were subcutaneously injected on the 28th day, 21st day, and 14th day before melanoma inoculation.
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。 In the experiment, the size of the mouse tumor volume was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. Due to the ethics of animal experiments, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图21所示，与PBS空白对照组，空白微米粒+细胞裂解物对照组相比，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，微米疫苗给药组中小鼠有部分小鼠肿瘤接种后完全消失。而且，肺癌细胞制备的微米疫苗较黑色素瘤细胞制备的微米疫苗取得了更好的预防效果。由此可见，本发明所述的负载肺癌细胞水溶性组分的微米疫苗对黑色素瘤具有交叉预防效果。(4) Experimental results: As shown in Figure 21, compared with the PBS blank control group and the blank microparticle + cell lysate control group, the growth rate of tumor volume in the micron vaccine administration group was significantly slower and the survival time of the mice was significantly slower. were significantly prolonged. Moreover, some tumors in mice in the micron vaccine administration group completely disappeared after inoculation. Moreover, the micron vaccine prepared by lung cancer cells achieved a better preventive effect than the micron vaccine prepared by melanoma cells. It can be seen that the micro-vaccine loaded with water-soluble components of lung cancer cells according to the present invention has a cross-preventive effect on melanoma.
实施例3 肺癌肿瘤组织裂解组分负载于纳米粒子内部和表面用于肝癌的预防：本实施例以如何制备负载有肺癌肿瘤组织裂解物组分的纳米疫苗，并应用该疫苗预防肝癌来说明如何使用肺癌肿瘤组织制备的疫苗交叉预防肝癌。Example 3 Lung cancer tumor tissue lysate components loaded inside and on the surface of nanoparticles for the prevention of liver cancer: This example describes how to prepare a nano-vaccine loaded with lung cancer tumor tissue lysate components and apply the vaccine to prevent liver cancer Cross-prophylaxis against liver cancer using a vaccine prepared from lung cancer tumor tissue.
本实施例中，将小鼠LLC肺癌肿瘤组织裂解组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠LLC肺癌肿瘤组织并将其裂解以制备肿瘤组织的水溶性组分和溶于8M尿素中的原非水溶性组分。然后，以PLGA 为纳米粒骨架材料，以poly(I:C)为免疫佐剂制备负载有裂解物的水溶性组分和非水溶性组分的纳米疫苗，并采用该疫苗来预防Hepa 1-6 肝癌。In this example, the lysed components of mouse LLC lung cancer tumor tissue were loaded inside and on the surface of nanoparticles to prepare nanovaccine. First, the mouse LLC lung cancer tumor tissue was obtained and lysed to prepare the water-soluble fraction of the tumor tissue and the original water-insoluble fraction dissolved in 8M urea. Then, using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant to prepare nanovaccine loaded with water-soluble components and non-water-soluble components of the lysate, and use the vaccine to prevent Hepatocellular carcinoma 1-6 Liver cancer.
（1）肿瘤组织的裂解及各组分的收集：方法同实施例1。(1) Lysis of tumor tissue and collection of components: the method is the same as in Example 1.
（2）纳米疫苗的制备：方法同实施例1。(2) Preparation of nano-vaccine: The method is the same as in Example 1.
（3）负载肺癌肿瘤组织的纳米疫苗用于肝癌的预防：选取6-8周的雌性C57BL/6制备 Hepa 1-6 肝癌荷瘤小鼠。(3) The nano-vaccine loaded with lung cancer tumor tissue is used for the prevention of liver cancer: select 6-8 week old female C57BL/6 to prepare Hepa 1-6 liver cancer tumor-bearing mice.
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内部和表面都负载组织裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子。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。 On days 49, 42, 35, 28, and 14 before inoculation of liver cancer cells, 200 μL of 2 mg PLGA nanoparticles loaded with water-soluble components in tissue lysates and 200 μL of internal and surface Both were loaded with 2mg of PLGA nanoparticles dissolved in 8M urea of the original water-insoluble component. On day 0, 2×10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
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400μL 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。 The protocol of the PBS blank control group was as follows: 400 μL of PBS was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. On day 0, 2×10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
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400μL 空白纳米粒和与疫苗所负载的等量的游离裂解物。空白纳米粒和游离组织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。 Blank nanoparticles + free lysate control group: Subcutaneously inject 400 μL of blank nanoparticles and the same amount of vaccine-loaded free lysate. Blank nanoparticles and free tissue lysates were injected at different sites. On day 0, 2×10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
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。 In the experiment, the size of the mouse tumor volume was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图22所示，PBS空白对照组以及空白纳米粒+组织裂解物对照组小鼠的肝癌肿瘤生长均较快。纳米疫苗给药组小鼠在接种肿瘤后肿瘤消失。由此可见，本发明所述的负载肺癌肿瘤组织裂解物中水溶性组分和非水溶性组分的纳米疫苗对肝癌具有交叉预防效果。(4) Experimental results: As shown in Figure 22, the liver cancer tumors in the PBS blank control group and the blank nanoparticle + tissue lysate control group grew faster. The tumors of the mice in the nanovaccine administration group disappeared after tumor inoculation. It can be seen that the nano-vaccine loaded with water-soluble components and water-insoluble components in the lung cancer tumor tissue lysate of the present invention has a cross-preventive effect on liver cancer.
实施例4 肺癌和黑色素瘤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，并应用该疫苗预防肝癌。Example 4 Whole cell components of lung cancer and melanoma tumor tissues loaded inside nanoparticles for the prevention of liver cancer: This example uses mouse liver cancer as a cancer model to illustrate how to prepare whole cell components loaded with lung cancer and melanoma tumor tissues Nano-vaccine, and apply the vaccine to prevent liver cancer.
本实施例中，首先裂解肺癌和黑色素瘤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。然后，以PLGA为纳米粒子骨架材料，以poly(I:C)为免疫佐剂采用溶剂挥发法制备同时负载有肺癌瘤块和黑色素瘤瘤块水溶性组分或非水溶性组分的纳米疫苗，并采用该纳米疫苗来预防肝癌。In this example, lung cancer and melanoma tumor tissues were first lysed to prepare the water-soluble and water-insoluble fractions of the whole cell fraction. Then, using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant, a nanovaccine loaded with water-soluble components or water-insoluble components of lung cancer tumor mass and melanoma tumor mass was prepared by solvent evaporation method. , and use the nano-vaccine to prevent liver cancer.
（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2×10 6个LLC肺癌细胞或接种1.5×10 5个B16F10黑色素瘤细胞，在肿瘤长到体积分别为约1000 mm 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与实施例1相同。 (1) Lysis of tumor tissue and collection of various components: Inoculate 2×10 6 LLC lung cancer cells or 1.5×10 5 B16F10 melanoma cells subcutaneously on the back of each C57BL/6 mouse, and inoculate them when the tumor grows to volume The mice were sacrificed when the diameter was about 1000 mm 3 , and the tumor tissues were removed. The lysing method of tumor tissue and the collection method of each component are the same as in Example 1.
（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7KDa-14KDa，所采用的免疫佐剂为 poly(I:C) 且poly(I:C)分布于纳米粒子内部。在制备疫苗时，水溶性组分为肺癌肿瘤组织水溶性组分和黑色素瘤肿瘤组织水溶性组分的混合物（等质量比）；非水溶性组分为肺癌肿瘤组织非水溶性组分和黑色素瘤肿瘤组织非水溶性组分的混合物（等质量比）。纳米疫苗粒径为300nm左右，纳米粒子平均表面电位为-6mV左右。每1 mg PLGA纳米粒子约负载200 μg蛋白质或多肽组分, 每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂为0.01mg。空白纳米粒粒径为240nm左右，空白纳米粒制备时分别采用含有poly(I:C）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，空白纳米粒子负载与纳米疫苗等量的poly(I:C)。(2) Preparation of nano-vaccine: In this embodiment, the preparation of nano-vaccine adopts the double emulsion method in the solvent evaporation method, the molecular weight of the nanoparticle preparation material PLGA used is 7KDa-14KDa, and the immune adjuvant used is poly(I:C) and poly(I:C) are distributed inside the nanoparticles. When preparing the vaccine, the water-soluble component is a mixture (equal mass ratio) of the water-soluble component of lung cancer tumor tissue and the water-soluble component of melanoma tumor tissue; the water-insoluble component is the water-insoluble component of lung cancer tumor tissue and melanin A mixture of tumor tissue water-insoluble components (equal mass ratio). The particle size of the nano-vaccine is about 300nm, and the average surface potential of the nano-particles is about -6mV. Each 1 mg of PLGA nanoparticles is loaded with about 200 μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside of each 1 mg of PLGA nanoparticles is 0.01 mg. The particle size of blank nanoparticles is about 240nm. When preparing blank nanoparticles, pure water containing poly(I:C) or 8M urea are used to replace the corresponding water-soluble components and non-water-soluble components. Blank nanoparticles are loaded with nano Vaccine equivalent poly(I:C).
（3）纳米疫苗用于癌症的预防：疫苗组和对照组具体给药方案和肿瘤生长监测方案如实施例3。(3) The nano-vaccine is used for the prevention of cancer: the specific administration scheme and tumor growth monitoring scheme of the vaccine group and the control group are as in Example 3.
（4）实验结果：如图23所示，与对照组相比，纳米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，疫苗组大部分小鼠肿瘤接种后消失。由此可见，本发明所述的负载肺癌肿瘤组织和黑色素瘤肿瘤组织裂解物中水溶性组分和非水溶性组分的纳米疫苗对肝癌具有预防效果。(4) Experimental results: As shown in Figure 23, compared with the control group, the tumor growth rate and the survival period of the mice in the nano-vaccine prevention group were significantly different. Moreover, most of the tumors in the mice in the vaccine group disappeared after inoculation. It can be seen that the nano-vaccine loaded with water-soluble components and water-insoluble components in the lysate of lung cancer tumor tissue and melanoma tumor tissue according to the present invention has a preventive effect on liver cancer.
实施例5 黑色素瘤肿瘤组织和结肠癌肿瘤组织裂解组分负载于纳米粒子内部和表面用于胰腺癌的治疗：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黑色素瘤肿瘤组织和结肠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。Example 5 Lysis components of melanoma tumor tissue and colon cancer tumor tissue are loaded inside and on the surface of nanoparticles for the treatment of pancreatic cancer: This example uses mouse pancreatic cancer as a cancer model to illustrate how to prepare melanoma tumor tissue and colon cancer tumor tissue lysate components, and apply the vaccine to treat pancreatic cancer.
本实施例中，将小鼠B16F10黑色素瘤肿瘤组织和MC38结肠癌肿瘤组织裂解组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠黑色素瘤和结肠癌肿瘤组织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。在制备疫苗时，水溶性组分为结肠癌肿瘤组织水溶性组分和黑色素瘤肿瘤组织水溶性组分的2:1质量比混合物；非水溶性组分为结肠癌肿瘤组织非水溶性组分和黑色素瘤肿瘤组织非水溶性组分的2:1质量比混合物。以PLGA为纳米粒子骨架材料，以poly(I:C)为免疫佐剂制备负载有肿瘤组织裂解物的水溶性组分和非水溶性组分的纳米疫苗。然后采用该疫苗治疗Pan02胰腺癌荷瘤小鼠。In this example, mouse B16F10 melanoma tumor tissue and MC38 colon cancer tumor tissue lysate components were loaded on the interior and surface of nanoparticles to prepare nanovaccine. First, mouse melanoma and colon cancer tumor tissues were obtained and lysed to prepare the water-soluble fraction and the original water-insoluble fraction dissolved in 8M urea. When preparing the vaccine, the water-soluble component is a 2:1 mass ratio mixture of the water-soluble component of the colon cancer tumor tissue and the water-soluble component of the melanoma tumor tissue; the water-insoluble component is the water-insoluble component of the colon cancer tumor tissue and a 2:1 mass ratio mixture of water-insoluble components of melanoma tumor tissue. Using PLGA as nanoparticle framework material and poly(I:C) as immune adjuvant, nanovaccine loaded with water-soluble and water-insoluble components of tumor tissue lysate was prepared. The vaccine was then used to treat Pan02 pancreatic cancer tumor-bearing mice.
（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2×10 6个MC38结肠癌细胞或接种1.5×10 5个B16F10黑色素瘤细胞，在肿瘤长到体积分别为约1000 mm 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与实施例1相同。 (1) Lysis of tumor tissue and collection of various components: subcutaneously inoculate 2×10 6 MC38 colon cancer cells or 1.5×10 5 B16F10 melanoma cells on the back of each C57BL/6 mouse, and inoculate when the tumor grows to The mice were sacrificed when the volume was about 1000 mm 3 and the tumor tissues were removed. The lysing method of tumor tissue and the collection method of each component are the same as in Example 1.
（2）纳米疫苗的制备：本实施例中制备纳米疫苗制备方法同实施例4。(2) Preparation of nano-vaccine: The preparation method of nano-vaccine in this example is the same as that in Example 4.
 （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200μL内部和表面都负载裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子。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400μL PBS。空白纳米粒+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400μL 空白纳米粒和与疫苗所负载的等量的游离裂解物。在实验中，从第3天起每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。 (3) Nano-vaccine for cancer treatment: select 6-8 week old female C57BL/6 to prepare pancreatic cancer tumor mice. On day 0, 1× 106 Pan02 cells were subcutaneously inoculated into the lower right lower back of each mouse. The vaccine group was subcutaneously injected with 200 μL of 2 mg PLGA nanoparticles loaded with water-soluble components in the lysate and 200 μL of lysate loaded with lysate on the 4th day, 7th day, 10th day, 15th day and 20th day, respectively. 2mg PLGA nanoparticles of the original water-insoluble component in 8M urea. The PBS blank control group was subcutaneously injected with 400 μL of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day. Blank nanoparticles + lysate control group were subcutaneously injected with 400 μL of blank nanoparticles and the same amount of free lysate loaded with the vaccine on the 4th day, 7th day, 10th day, 15th day and 20th day. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图24所示，与对照组相比，纳米疫苗治疗组肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，有部分小鼠肿瘤接种后消失。由此可见，本发明所述的负载黑色素瘤和结肠癌肿瘤组织裂解物中水溶性组分和非水溶性组分的纳米疫苗对胰腺癌具有交叉治疗效果。(4) Experimental results: As shown in Figure 24, compared with the control group, the tumor growth rate of the nano-vaccine treatment group was significantly slower and the survival period of the mice was significantly prolonged. Moreover, tumors in some mice disappeared after inoculation. It can be seen that the nano-vaccine loaded with water-soluble components and water-insoluble components in the tumor tissue lysates of melanoma and colon cancer described in the present invention has a cross-therapeutic effect on pancreatic cancer.
实施例 6 黑色素瘤肿瘤组织全细胞组分负载于微米粒子内部用于肺癌的预防：本实施例以小鼠肺癌为癌症模型来说明如何制备负载有黑色素瘤肿瘤组织全细胞组分的微米疫苗，并应用该疫苗预防肺癌。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。Example 6 Whole cell components of melanoma tumor tissue loaded inside microparticles for the prevention of lung cancer: This example uses mouse lung cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with whole cell components of melanoma tumor tissue, And apply the vaccine to prevent lung cancer. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
本实施例中，将小鼠黑色素瘤肿瘤组织裂解组分负载于微米粒子内部以制备微米疫苗。首先取得小鼠黑色素瘤肿瘤组织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(50:50) 和甘露糖修饰的PLGA为微米粒骨架材料，以CpG为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织裂解物的水溶性组分和非水溶性组分的微米疫苗。该微米疫苗具有靶向树突状细胞的能力。In this example, the lysed components of mouse melanoma tumor tissue were loaded inside the microparticles to prepare micron vaccines. First, mouse melanoma tumor tissues were obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea. Then, PLGA (50:50) and mannose-modified PLGA were used as the microparticle framework material, and CpG was used as the immune adjuvant to prepare the water-soluble and non-water-soluble components loaded with tumor tissue lysates by the solvent evaporation method. micron vaccine. The micron vaccine has the ability to target dendritic cells.
（1）肿瘤组织的裂解及各组分的收集：在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16F10黑色素瘤细胞，在小鼠所接种肿瘤长到1000 mm 3时处死小鼠并摘取肿瘤组织。肿瘤组织裂解和组分收集方法同实施例1。 (1) Lysis of tumor tissue and collection of various components: 1.5× 105 B16F10 melanoma cells were subcutaneously inoculated on the back of each C57BL/6 mouse, and the mice were killed when the inoculated tumor grew to 1000 mm3 And remove the tumor tissue. The method of lysing tumor tissue and collecting components is the same as that in Example 1.
（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溶剂挥发法中的复乳法，所采用微米粒子制备材料PLGA(50:50) 分子量为38KDa-54KDa，所采用的甘露糖修饰的PLGA(50:50)分子量为38KDa-54KDa。在靶头修饰微米疫苗组中未修饰PLGA，甘露糖修饰的PLGA的质量比为8: 2。无靶头修饰微米疫苗组全部采用未修饰PLGA制备。所采用的免疫佐剂为CpG且CpG分布于微米粒子内部。制备方法如前所述微米粒子平均粒径为1.20μm左右，平均表面电位为 -8 mV左右。每1 mg PLGA微米粒子负载 60μg 蛋白质或多肽组分, 每 1mg PLGA微米粒内外所使用的CpG免疫佐剂为0.01mg且内外各半。空白微米粒粒径为1.10 μm 左右，空白微米粒制备时分别采用含有等量CpG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。(2) Preparation of micron vaccines: In this example, the micron vaccines and the empty micron particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micron particle preparation material PLGA (50:50) is used The molecular weight is 38KDa-54KDa, and the molecular weight of the mannose-modified PLGA (50:50) used is 38KDa-54KDa. In the target modified micron vaccine group, the mass ratio of unmodified PLGA to mannose-modified PLGA was 8:2. The non-target modified micro-vaccine group was all prepared by unmodified PLGA. The immune adjuvant used is CpG and the CpG is distributed inside the microparticles. The preparation method is as mentioned above. The average particle size of the micron particles is about 1.20 μm, and the average surface potential is about -8 mV. per 1 mg PLGA microparticles are loaded with 60 μg of protein or polypeptide components, and the CpG immune adjuvant used for each 1 mg of PLGA microparticles is 0.01 mg, and the inside and outside are divided in half. The particle size of the blank microparticles is about 1.10 μm. When the blank microparticles are prepared, pure water or 8M urea containing the same amount of CpG is used to replace the corresponding water-soluble components and non-water-soluble components.
（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C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL内部和表面都负载癌细胞裂解物中水溶性成分的2mg PLGA微米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA微米粒子。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400μL PBS。空白微米粒+细胞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400μL 空白微米粒和与疫苗所负载的等量的游离细胞裂解物。在第0天给每只小鼠背部右下方皮下接种2×10 6个个LLC肺癌细胞。在实验中，从第3天起每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。 (3) Micro-vaccine targeting dendritic cells for the prevention of cancer: select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice. On the 35th, 28th, 21st, 14th, and 7th days before tumor inoculation, the vaccine group received subcutaneous injections of 200 μL of 2 mg PLGA microparticles loaded with water-soluble components in cancer cell lysates and 200 μL of internal Both the surface and the surface are loaded with 2mg PLGA micron particles dissolved in 8M urea, which is the original water-insoluble component. The PBS blank control group was subcutaneously injected with 400 μL PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. Blank microparticles + cell lysate control group were subcutaneously injected with 400 μL of blank microparticles and the same amount of free cells loaded with the vaccine on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. Lysate. On day 0, 2×10 6 LLC lung cancer cells were subcutaneously inoculated into the lower right lower back of each mouse. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图25所示，与PBS空白对照组和空白微米粒+细胞裂解物对照组相比，微米疫苗组小鼠肿瘤生长速度明显变慢且小鼠生存期明显延长。这说明本发明所述的负载黑色素瘤肿瘤组织中水溶性组分和非水溶性组分主动靶向微米疫苗肺癌具有预防效果，而且靶头修饰的微米疫苗(Mannose modified)要好于未经靶头修饰的微米疫苗。(4) Experimental results: As shown in Figure 25, compared with the PBS blank control group and the blank microparticle + cell lysate control group, the tumor growth rate of the mice in the micron vaccine group was significantly slower and the survival period of the mice was significantly prolonged. This shows that the water-soluble components and non-water-soluble components in the melanoma tumor tissue of the present invention actively target the micron vaccine to prevent lung cancer, and the target-modified micron vaccine (Mannose modified) is better than micron vaccines without target modification.
实施例7 肺癌肿瘤组织或黑色素瘤肿瘤组织全细胞组分负载于纳米粒子内部和表面并以卡介苗（BCG）为免疫佐剂的纳米疫苗用于肝癌的预防：本实施例以小鼠肝癌为癌症模型并以BCG为免疫佐剂来说明如何采用负载有肺癌肿瘤组织或者黑色素瘤肿瘤组织全细胞组分的纳米疫苗预防肝癌。Example 7 The whole cell components of lung cancer tumor tissue or melanoma tumor tissue are loaded inside and on the surface of nanoparticles, and the nano-vaccine with bacillus Calmette-Guerin (BCG) as an immune adjuvant is used for the prevention of liver cancer: this example takes mouse liver cancer as the cancer Model and use BCG as an immune adjuvant to illustrate how to use nano-vaccine loaded with whole cell components of lung cancer tumor tissue or melanoma tumor tissue to prevent liver cancer.
本实施例中，首先裂解肺癌或黑色素瘤肿瘤组织的水溶性组分和非水溶性组分。然后，以PLGA为纳米粒子骨架材料，以BCG为免疫佐剂分别制备负载有肺癌或者黑色素瘤肿瘤组织水溶性组分和非水溶性组分的纳米疫苗。In this embodiment, first, the water-soluble and water-insoluble components of lung cancer or melanoma tumor tissue are lysed. Then, using PLGA as the nanoparticle framework material and BCG as the immune adjuvant to prepare nanovaccine loaded with water-soluble components and water-insoluble components of lung cancer or melanoma tumor tissue.
（1）肿瘤组织的裂解及各组分的收集：该实施例中肿瘤组织裂解及裂解物收集和增溶方法同实施例1。(1) Lysis of tumor tissue and collection of various components: In this embodiment, the method of lysing tumor tissue and collecting and solubilizing the lysate is the same as that in Example 1.
（2）BCG的裂解及各组分的收集：该实施例中BCG的裂解及裂解物收集和增溶方法同实施例2中癌细胞的裂解方法，只是将癌细胞换成BCG。(2) Lysis of BCG and collection of various components: The method of lysing BCG and collecting and solubilizing lysates in this example is the same as that of cancer cells in Example 2, except that the cancer cells are replaced with BCG.
（3）纳米疫苗的制备：本实施例中纳米疫苗的制备方法、所使用的材料等均与实施例1相同。但是在该实施例中，纳米疫苗负载的免疫佐剂由poly(I:C)换成了BCG裂解物中的水溶性成分或非水溶性成分。(3) Preparation of nano-vaccine: The preparation method and materials used in this example are the same as those in Example 1. However, in this example, the immune adjuvant loaded by the nanovaccine is replaced by poly(I:C) water-soluble or water-insoluble components in BCG lysates.
（4）纳米疫苗用于肝癌的预防：选取雌性C57BL/6为模型小鼠制备Hepa 1-6 肝癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL内部和表面都负载肿瘤组织裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米疫苗。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400μL PBS。空白纳米粒+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400μL 空白纳米粒和与疫苗所负载的等量的游离裂解物。在第0天给每只小鼠腋下皮下接种2×10 6个Hepa1-6肝癌细胞。在实验中，从第3天起每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) Nano-vaccine for the prevention of liver cancer: Select female C57BL/6 as model mice to prepare Hepa 1-6 liver cancer tumor-bearing mice. The vaccine group was subcutaneously injected with 200 μL of 2 mg PLGA nanoparticles loaded with water-soluble components in tumor tissue lysate and 200 μL of the internal surface on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation, respectively. Both the surface and the surface are loaded with the 2mg PLGA nano-vaccine dissolved in the original water-insoluble component in 8M urea. The PBS blank control group was subcutaneously injected with 400 μL PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. Blank nanoparticles + lysate control group were subcutaneously injected with 400 μL of blank nanoparticles and the same amount of free lysate loaded with the vaccine on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation. . On day 0, 2× 106 Hepa1-6 liver cancer cells were subcutaneously inoculated into the armpit of each mouse. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图26所示，与对照组相比，以BCG为佐剂的纳米疫苗给药组肿瘤生长速度明显变慢且小鼠生存期明显延长。由此可见，本发明所述负载肺癌或者黑色素瘤肿瘤组织全细胞组分的纳米疫苗可以预防肝癌。而且，负载肺癌肿瘤组织全细胞组分的纳米疫苗对肝癌交叉预防的效果要好于负载黑色素瘤肿瘤组织全细胞组分的纳米疫苗。(4) Experimental results: As shown in Figure 26, compared with the control group, the tumor growth rate of the nanovaccine administration group with BCG as adjuvant was significantly slower and the survival period of the mice was significantly prolonged. It can be seen that the nano-vaccine loaded with whole cell components of lung cancer or melanoma tumor tissue in the present invention can prevent liver cancer. Moreover, the nanovaccine loaded with whole cell components of lung cancer tumor tissue has a better cross-prevention effect on liver cancer than the nanovaccine loaded with whole cell components of melanoma tumor tissue.
实施例8 6M盐酸胍溶解肺癌和结肠癌肿瘤组织组分并负载于微米粒子内部和表面用于乳腺癌的治疗：本实施例以小鼠乳腺癌为癌症模型来说明如何采用6M盐酸胍溶解全细胞组分并制备负载有全细胞组分的微米疫苗以治疗乳腺癌。本实施例中，以4T1小鼠三阴性乳腺癌细胞为癌细胞模型。首先对肺癌和结肠癌肿瘤组织细胞进行灭活和变性处理并以6M盐酸胍裂解肿瘤组织并溶解全细胞组分。然后，以PLGA为微米粒子骨架材料，以CpG为免疫佐剂制备负载全细胞组分的微米疫苗。然后采用该微米疫苗来治疗乳腺癌荷瘤小鼠体内的肿瘤。Example 8 6M guanidine hydrochloride dissolves tumor tissue components of lung cancer and colon cancer and loads them inside and on the surface of microparticles for the treatment of breast cancer: This example uses mouse breast cancer as a cancer model to illustrate how to use 6M guanidine hydrochloride to dissolve whole Cell components and preparation of micron vaccines loaded with whole cell components for the treatment of breast cancer. In this example, 4T1 mouse triple-negative breast cancer cells were used as cancer cell models. Firstly, the tumor tissue cells of lung cancer and colon cancer were inactivated and denatured, and the tumor tissue was lysed with 6M guanidine hydrochloride to dissolve the whole cell components. Then, PLGA is used as the framework material of micron particles, and CpG is used as immune adjuvant to prepare the micron vaccine loaded with whole cell components. The microvaccine was then used to treat tumors in breast cancer-bearing mice.
（1）肿瘤组织的裂解及各组分的收集：在C57BL/6小鼠右腋下皮下接种2×10 6个LLC肺癌细胞或者接种2×10 6个MC38结肠癌细胞，在肿瘤长到体积1000 mm 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6M盐酸胍裂解肺癌和结肠癌肿瘤组织细胞并溶解组织裂解物，将肺癌的肿瘤组织裂解物与结肠癌肿瘤组织裂解物混合后即为制备疫苗的原料来源。 (1) Lysis of tumor tissue and collection of various components: Inoculate 2×10 6 LLC lung cancer cells or 2×10 6 MC38 colon cancer cells subcutaneously in the right armpit of C57BL/6 mice, and when the tumor grows to volume Mice were sacrificed at 1000 mm 3 and tumor tissues were removed. Cut the tumor tissue into pieces, grind it, filter it through a cell strainer, and collect the tumor tissue cells obtained by filtering. The obtained tumor tissue cells were inactivated and denatured by ultraviolet light and high temperature heating respectively, and then the tumor tissue cells of lung cancer and colon cancer were lysed by appropriate amount of 6M guanidine hydrochloride and the tissue lysate was dissolved, and the tumor tissue lysate of lung cancer and colon cancer tumor tissue were lysed After the mixture is mixed, it is the source of raw materials for preparing vaccines.
（2）微米疫苗的制备：本实施例中微米疫苗及空白微米粒子采用分子量为38KD-54KD的PLGA（50:50），制备方法如前所述。 采用CpG为免疫佐剂。所制备微米疫苗平均粒径为2.5μm左右，微米粒子表面电位为-4mV。每1mg PLGA微米粒子内外负载蛋白质和多肽组分为210μg，每1mgPLGA纳米粒内外所使用的CpG免疫佐剂共0.01mg且内外各半。(2) Preparation of micron vaccine: In this example, the micron vaccine and blank micron particles used PLGA (50:50) with a molecular weight of 38KD-54KD, and the preparation method was as described above. CpG was used as an immune adjuvant. The average particle size of the prepared micron vaccine is about 2.5 μm, and the surface potential of the micron particle is -4mV. 210 μg of protein and polypeptide components are loaded inside and outside each 1 mg PLGA nanoparticle, and the CpG immune adjuvant used inside and outside each 1 mg PLGA nanoparticle is 0.01 mg in total, and the inside and outside are divided into half.
（3）微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400μL内部和表面都负载肿瘤组织全细胞组分的4 mg PLGA微米疫苗。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400μL PBS。空白微米粒+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，以及负载有等量CpG而不负载任何细胞裂解物成分的4mg PLGA空白微米粒。在实验中，从第3天起每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。生存期实验中小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。 (3) Micron vaccine for the treatment of cancer: Select 6-8 weeks old female BALB/c to prepare 4T1 tumor-bearing mice. On day 0, 4 × 105 4T1 cells were subcutaneously inoculated into the lower right lower back of each mouse. The vaccine treatment group was subcutaneously injected with 400 μL of 4 mg PLGA micron vaccine loaded with whole cell components of tumor tissue inside and on the surface on the 4th, 7th, 10th, 15th and 20th days. The PBS blank control group was subcutaneously injected with 400 μL of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day. Blank microparticles + lysate control group were subcutaneously injected with equal amount of tumor tissue lysate on the 4th day, 7th day, 10th day, 15th day and 20th day, and loaded with the same amount of CpG without any cell lysis 4mg PLGA blank micron particles of the material composition. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. In the survival experiment, mice whose tumor volume exceeded 2000 mm3 were considered dead and were euthanized.
（4）实验结果：如图27所示，与对照组相比，负载两种肿瘤组织全细胞组分的微米疫苗给药组肿瘤生长速度明显变慢且小鼠生存期明显延长。由此可见，本发明所述的负载肺癌和结肠癌肿瘤组织全细胞组分的微米疫苗对乳腺癌具有治疗效果。(4) Experimental results: As shown in Figure 27, compared with the control group, the growth rate of the micron vaccine administered with the whole cell components of two tumor tissues was significantly slower and the survival period of the mice was significantly prolonged in the administration group. It can be seen that the micro-vaccine loaded with whole cell components of lung cancer and colon cancer tumor tissue of the present invention has a therapeutic effect on breast cancer.
实施例9 黑色素瘤肿瘤组织裂解组分负载于纳米粒子内部和表面用于肝癌的预防：本实施例以如何制备负载有黑色素瘤肿瘤组织裂解物组分的纳米疫苗，并应用该疫苗预防肝癌来说明如何使用黑色素瘤肿瘤组织制备的疫苗交叉预防肝癌。Example 9 Melanoma tumor tissue lysate components are loaded inside and on the surface of nanoparticles for the prevention of liver cancer: This example is based on how to prepare a nano-vaccine loaded with melanoma tumor tissue lysate components and apply the vaccine to prevent liver cancer To illustrate how to use a vaccine prepared from melanoma tumor tissue for cross-prevention of liver cancer.
本实施例中，将小鼠B16F10黑色素瘤肿瘤组织裂解组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠肿瘤组织并将其裂解以制备肿瘤组织的水溶性组分和溶于6M盐酸胍中的原非水溶性组分。然后，以PLGA 为纳米粒骨架材料，以poly(I:C)为免疫佐剂或不带免疫佐剂制备负载有裂解物的水溶性组分和非水溶性组分的纳米疫苗，并采用该疫苗来预防Hepa 1-6 肝癌。In this example, mouse B16F10 melanoma tumor tissue lysate components were loaded on the inside and surface of nanoparticles to prepare nanovaccine. First, mouse tumor tissue was obtained and lysed to prepare the water-soluble fraction of the tumor tissue and the original water-insoluble fraction dissolved in 6M guanidine hydrochloride. Then, using PLGA as the nanoparticle framework material, using poly(I:C) as an immune adjuvant or without an immune adjuvant to prepare nanovaccine loaded with water-soluble and water-insoluble components of the lysate, and using the Vaccine to prevent Hepa 1-6 liver cancer.
（1）肿瘤组织的裂解及各组分的收集：方法同实施例1。(1) Lysis of tumor tissue and collection of components: the method is the same as in Example 1.
（2）纳米疫苗的制备：方法同实施例1，不含免疫佐剂组不加入poly(I:C) 。(2) Preparation of nano-vaccine: The method is the same as in Example 1, except that poly(I:C) is not added to the group without immune adjuvant.
（3）负载肿瘤组织的纳米疫苗用于肝癌的预防：选取6-8周的雌性C57BL/6制备 Hepa 1-6 肝癌荷瘤小鼠。(3) Nanovaccine loaded with tumor tissue is used for the prevention of liver cancer: 6-8 week old female C57BL/6 were selected to prepare Hepa 1-6 liver cancer tumor-bearing mice.
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内部和表面都负载肿瘤组织裂解物中水溶性成分的2mg PLGA纳米疫苗和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA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。 On the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells, 200 μL of 2 mg PLGA nano-vaccine loaded with water-soluble components in tumor tissue lysates and 200 μL of internal and The surfaces are all loaded with 2 mg PLGA nano-vaccine dissolved in 8M urea, which is the original water-insoluble component. On day 0, 2×10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
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400μL 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。 The protocol of the PBS blank control group was as follows: 400 μL of PBS was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. On day 0, 2×10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
空白纳米粒+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400μL 空白纳米粒和与疫苗所负载的等量的游离细胞裂解物。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。 Blank nanoparticles + lysate control group: Subcutaneously inject 400 μL of blank nanoparticles and free cell lysate. Blank nanoparticles and free cell lysates were injected at different sites. On day 0, 2×10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse.
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。 In the experiment, the size of the mouse tumor volume was recorded every 3 days from the 3rd day. Tumor volume was calculated using the formula v=0.52×a× b2 , where v was the tumor volume, a was the tumor length, and b was the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeds 2000mm3 , the mouse is considered dead and the mouse is euthanized.
（4）实验结果：如图28所示，PBS空白对照组以及空白纳米粒+组织裂解物对照组小鼠的肝癌肿瘤生长均较快。纳米疫苗给药组小鼠在接种肿瘤后部分小鼠肿瘤消失。而且，含有免疫佐剂的疫苗组小鼠肿瘤生长速度明显慢于不含佐剂的疫苗组。这说明免疫佐剂有助于疫苗发挥功效。(4) Experimental results: As shown in Figure 28, the liver cancer tumors in the PBS blank control group and the blank nanoparticle + tissue lysate control group grew faster. The tumors of mice in the nanovaccine administration group disappeared after tumor inoculation. Moreover, the tumor growth rate of mice in the vaccine group containing immune adjuvant was significantly slower than that in the vaccine group without adjuvant. This shows that the immune adjuvant helps the vaccine to play a role.
实施例10肺癌癌细胞全细胞组分负载于纳米粒子内部和表面用于黑色素瘤的预防：本实施例说明如何制备负载有肺癌癌细胞的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，首先裂解LLC癌细胞以制备相应的水溶性组分和溶于8M尿素的非水溶性组分。然后，以PLGA为骨架材料，以胶体锰为免疫佐剂制备纳米疫苗。Example 10 Whole cell components of lung cancer cancer cells are loaded on the inside and surface of nanoparticles for the prevention of melanoma: This example illustrates how to prepare a nano-vaccine loaded with whole cell components of lung cancer cancer cells, and apply the vaccine to prevent melanoma tumor. In this example, LLC cancer cells were first lysed to prepare the corresponding water-soluble components and water-insoluble components dissolved in 8M urea. Then, using PLGA as the framework material and colloidal manganese as the immune adjuvant to prepare the nano-vaccine.
（1）癌细胞的裂解及各组分的收集：LLC肺癌癌细胞的裂解方法及各组分收集方法同上。(1) Lysis of cancer cells and collection of various components: The lysis method of LLC lung cancer cells and the collection of various components are the same as above.
（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复乳法制备，水溶性组分负载于纳米粒子内部而非水溶性组分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胶体锰且胶体锰分布于纳米粒子内部。在180μL Na 3PO 4 (0.028M)中加入20μLMnCl 2 (0.2M)制得胶体锰，然后与300μL水溶性组分（80 mg/mL）混合后加入1 mL含100mg PLGA的二氯甲烷中超声制备初乳，尔后将上述样品加入2.5 mL 的20 mg/mL 聚乙烯醇（PVA）水溶液中超声，尔后加入50 mL含5 mg/mL PVA的水溶液中搅拌3小时，尔后在12000g离心30 min 后采用10 mL 4%的海藻糖水溶液重悬纳米粒并冷冻干燥48h。在使用前将上述样品溶于9 mL PBS中并于1 mL 溶于8M尿素中的非水溶性组分（80mg/mL）混合后室温作用10 min后即可使用。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1 mg PLGA纳米粒子约负载180μg蛋白质或多肽组分。空白纳米粒粒径为320nm左右，空白纳米粒负载等量的胶体锰。 (2) Preparation of nano-vaccine: In this example, the nano-vaccine and the blank nano-particles were prepared by the double emulsion method, and the water-soluble components were loaded inside the nanoparticles instead of the water-soluble components on the surface of the nano-vaccine. The nanoparticles used The molecular weight of the prepared material PLGA is 7KDa-17KDa, the immune adjuvant adopted is colloidal manganese and the colloidal manganese is distributed inside the nanoparticles. Add 20 μL MnCl 2 (0.2M) to 180 μL Na 3 PO 4 (0.028M) to prepare colloidal manganese, then mix with 300 μL water-soluble fraction (80 mg/mL) and add 1 mL of dichloromethane containing 100 mg PLGA to sonicate Prepare colostrum, then add the above sample into 2.5 mL of 20 mg/mL polyvinyl alcohol (PVA) aqueous solution for ultrasonication, then add 50 mL of 5 mg/mL PVA aqueous solution and stir for 3 hours, then centrifuge at 12000g for 30 min Nanoparticles were resuspended in 10 mL of 4% trehalose aqueous solution and freeze-dried for 48 h. Before use, the above samples were dissolved in 9 mL of PBS and mixed with 1 mL of non-water-soluble components (80 mg/mL) dissolved in 8M urea, and then used at room temperature for 10 min. The average particle size of the nano-vaccine loaded with whole cell components is about 350nm, and the surface potential of the nano-vaccine is about -5mV; each 1 mg PLGA nano-particle is loaded with about 180 μg protein or polypeptide component. The particle size of the blank nanoparticles is about 320nm, and the blank nanoparticles are loaded with an equal amount of colloidal manganese.
（3）纳米疫苗用于癌症的预防：疫苗组小鼠每次每只小鼠给药药剂量为400μL 含4 mg PLGA 的纳米疫苗，对照组组每次每只小鼠给予400 μL PBS或者空白纳米粒+游离裂解物。预防时的给药方案和小鼠肿瘤接种和监测方案实施例1。(3) Nano-vaccine for the prevention of cancer: mice in the vaccine group were given 400 μL of nano-vaccine containing 4 mg PLGA each time, and mice in the control group were given 400 μL of PBS or blank Nanoparticles + free lysate. Dosing regimen for prophylaxis and protocol for mouse tumor inoculation and monitoring Example 1.
（4）实验结果：如图29所示，疫苗处理组约90%小鼠的肿瘤在接种后消失；而PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大且生长速度很快。综上所述，负载肺癌癌细胞全细胞组分的纳米疫苗对黑色素瘤具有预防效果。(4) Experimental results: As shown in Figure 29, about 90% of the tumors of the mice in the vaccine treatment group disappeared after inoculation; while the tumors of the mice in the PBS control group and the blank nanoparticle control group grew and grew rapidly. In summary, nanovaccine loaded with whole cell components of lung cancer cells has a preventive effect on melanoma.
本发明所述全细胞组分的疫苗系统可用于制备交叉预防和/或治疗癌症的药物，其制备过程及应用领域如图30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增溶剂的增溶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。The vaccine system with whole cell components of the present invention can be used to prepare drugs for cross-prevention and/or treatment of cancer, and its preparation process and application fields are shown in FIG. 30 . During the preparation, the cells or tissues can be lysed, and then the water-soluble components and water-insoluble components can be collected separately to prepare nano-vaccine or micro-vaccine respectively; or directly use a solubilizing solution containing a solubilizing agent to directly lyse cells or tissues and dissolve whole cells Components and preparation of nano-vaccine or micro-vaccine.

Claims (10)

  1. 基于全细胞组分的纳米癌症疫苗和/或微米癌症疫苗系统在制备交叉预防或治疗异种癌症药物中的应用，其特征在于，所述基于全细胞组分的癌症疫苗系统包括全细胞组分、纳米和/或微米粒子；所述全细胞组分为癌细胞全细胞组分和/或肿瘤组织全细胞组分。The application of nano cancer vaccine and/or micron cancer vaccine system based on whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs, characterized in that the cancer vaccine system based on whole cell components includes whole cell components, Nano and/or micro particles; the whole cell component is the whole cell component of cancer cells and/or the whole cell component of tumor tissue.
  2. 根据权利要求1所述的应用，其特征在于，所述基于全细胞组分的癌症疫苗系统还包括免疫增强佐剂。The application according to claim 1, characterized in that the cancer vaccine system based on whole cell components further includes an immune enhancing adjuvant.
  3. 根据权利要求1所述的应用，其特征在于，所述纳米和/或微米粒子的制备材料包括有机合成高分子材料、天然高分子材料或者无机材料；所述纳米癌症疫苗的粒径为1nm～1000nm；所述微米癌症疫苗的粒径为1μm～1000μm；所述纳米粒子的粒径为1nm～1000nm；所述微米粒子的粒径为1μm～1000μm。The application according to claim 1, wherein the preparation materials of the nano and/or micro particles include organic synthetic polymer materials, natural polymer materials or inorganic materials; the particle diameter of the nano cancer vaccine is 1nm～ 1000 nm; the particle size of the micron cancer vaccine is 1 μm˜1000 μm; the particle size of the nanoparticle is 1 nm˜1000 nm; the particle size of the micron particle is 1 μm˜1000 μm.
  4. 根据权利要求1所述的应用，其特征在于，所述全细胞组分来源于一种或者多种实体瘤癌症或者非实体瘤癌症的癌细胞和/或肿瘤组织；所述交叉预防或治疗的异种癌症为不同于制备疫苗的癌细胞或肿瘤组织的癌症；所述异种癌症为一种或多种实体瘤癌症或者非实体瘤癌症。The application according to claim 1, wherein the whole cell components are derived from cancer cells and/or tumor tissues of one or more solid tumor cancers or non-solid tumor cancers; the cross-prevention or treatment A xenogeneic cancer is a cancer that is different from the cancer cells or tumor tissue from which the vaccine is made; the xenogeneic cancer is one or more solid tumor cancers or non-solid tumor cancers.
  5. 根据权利要求1所述的应用，其特征在于，所述全细胞组分为水溶性成份和/或非水溶性成份。The application according to claim 1, characterized in that, the whole cell component is a water-soluble component and/or a water-insoluble component.
  6. 根据权利要求5所述的应用，其特征在于，所述全细胞的水溶性成分为癌细胞或肿瘤组织中全细胞的可溶于纯水或不含增溶剂的水溶液中的原水溶性部分；所述全细胞的非水溶性成分为癌细胞或肿瘤组织中全细胞的原非水溶性部分采用增溶方法由在纯水中不溶变为在含增溶剂的水溶液中或有机溶剂中可溶的部分。The application according to claim 5, characterized in that, the water-soluble component of the whole cell is the original water-soluble part of the whole cell in cancer cells or tumor tissue that is soluble in pure water or an aqueous solution without a solubilizer; The water-insoluble component of the whole cell is the original water-insoluble part of the whole cell in the cancer cell or tumor tissue, which is changed from being insoluble in pure water to being soluble in an aqueous solution containing a solubilizing agent or in an organic solvent by solubilization method .
  7. 根据权利要求5所述的应用，其特征在于，全细胞的水溶性成分和/或非水溶性成分分别或同时被包载于纳米和/或微米粒子内部，和/或分别或同时负载于纳米和/或微米粒子表面。The application according to claim 5, characterized in that, the water-soluble components and/or water-insoluble components of the whole cells are respectively or simultaneously loaded inside the nanometer and/or microparticles, and/or are separately or simultaneously loaded on the nanoparticle and/or microparticle surfaces.
  8. 根据权利要求1所述的应用，其特征在于，所述基于全细胞组分的癌症疫苗系统表面可以不连接具有主动靶向功能的靶头或者连接有主动靶向功能的靶头。The application according to claim 1, characterized in that the surface of the whole cell component-based cancer vaccine system may not be connected with a target head with active targeting function or be connected with a target head with active targeting function.
  9. 一种交叉预防或治疗异种癌症的疫苗系统，包括癌细胞或肿瘤组织全细胞组分、纳米和/或微米粒子，其特征在于，所述疫苗系统用于交叉预防或交叉治疗不同于制备疫苗所使用的癌细胞或肿瘤组织的其他类型癌症。A vaccine system for cross-prevention or treatment of heterogeneous cancers, comprising whole cell components of cancer cells or tumor tissues, nanometers and/or microparticles, characterized in that the vaccine system used for cross-prevention or cross-treatment is different from that used for preparing vaccines Use of cancer cells or other types of tumor tissue.
  10. 全细胞组分在制备交叉预防或治疗异种癌症药物中的应用，其特征在于，所述全细胞组分为癌细胞全细胞组分和/或肿瘤组织全细胞组分。The application of the whole cell components in the preparation of cross-prevention or treatment of heterogeneous cancer drugs is characterized in that the whole cell components are cancer cell whole cell components and/or tumor tissue whole cell components.
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