WO2023040121A1 - Vaccine system for preventing or treating cancer on the basis of multiple cancer cells and/or tumor tissue whole cell components, and preparation therefor and application thereof - Google Patents
Vaccine system for preventing or treating cancer on the basis of multiple cancer cells and/or tumor tissue whole cell components, and preparation therefor and application thereof Download PDFInfo
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- WO2023040121A1 WO2023040121A1 PCT/CN2021/142691 CN2021142691W WO2023040121A1 WO 2023040121 A1 WO2023040121 A1 WO 2023040121A1 CN 2021142691 W CN2021142691 W CN 2021142691W WO 2023040121 A1 WO2023040121 A1 WO 2023040121A1
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Classifications
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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 nano or micro cancer vaccine based on different cancer cells and/or different tumor tissues, in particular to a whole-body vaccine based on two or more cancer cells and/or tumor tissues Cancer vaccines of cellular components and their use in the prevention and treatment 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 removing abnormal substances (such as viruses, bacteria, etc.) in the human body, or damaged cells produced by the human body itself and tumor cells to maintain human health.
- Immunotechnology has developed 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 balance of the body's immune system, it is expected to affect and control the occurrence, development and treatment of cancer.
- Cancer vaccines are one of the important approaches in cancer immunotherapy and prevention.
- 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.
- the object of the present invention is to solve the problems existing in the prior art, and provide a method for preventing or treating cancer with a micro or nano vaccine system loaded with whole cell components of various cancer cells and/or tumor tissues.
- the present invention adopts the following technical scheme: a nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components, including nano and/or micro Particles, a mixture of whole cell components; the mixture of whole cell components is whole cell components of various cancer cells and/or tumor tissues.
- the vaccine system for the prevention or treatment of cancer based on a variety of cancer cells and/or tumor tissue whole cell components is a nano or micro vaccine system, called a nano vaccine or a micro vaccine, which can prevent or treat cancer. or a mixture of micron-sized particles and said particle-loaded whole cell components, or a mixture of nano-sized or micron-sized particles and said particle-loaded whole cell components, and an immune adjuvant; said whole
- the cell component is a mixture of water-soluble components and/or a mixture of water-insoluble components of whole cells in different cancer cells and/or different tumor tissues.
- the mixture can be, but is not limited to, water soluble ingredients mixed with each other, or water insoluble ingredients mixed with each other, or all and/or partially water soluble components mixed with all and/or partially water soluble components.
- the preparation method of the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention is: Loaded inside and/or on the surface of nano and/or micro particles to obtain the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components; Cells and/or tumor tissue whole cell components and immune adjuvants are loaded inside and/or on the surface of nanometer and/or micron particles to obtain the prevention or treatment of cancer based on a variety of cancer cells and/or tumor tissue whole cell components nano and/or micro vaccine systems.
- the vaccine system for preventing or treating cancer based on a variety of cancer cells and/or tumor tissue whole cell components of the present invention can be prepared according to the preparation methods developed for nano-sized particles and micron-sized particles, including but not limited to Common solvent evaporation method, dialysis method, extrusion method, hot melt method.
- the vaccine system is prepared by the double emulsion method in the solvent evaporation method.
- the active ingredient whole cell component mixture of the nanometer and/or micron vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention is a water-soluble component mixture of whole cells and/or
- a mixture of water-insoluble components, prepared from two or more cancer cells and/or tumor tissues can be prepared from two or more cancer cells, or can be prepared from two or more tumor tissues, It can also be prepared from more than one kind of cancer cells and tumor tissues. This is where the creativity of the present invention lies.
- one kind of cancer cell or tumor tissue is used to prepare whole cell components for nano and/or micro particle construction of cancer vaccines.
- the present invention uses two or more cancer cells and/or Or tumor tissue to prepare cancer vaccines, unexpectedly achieved significant progress in technical effects.
- the nano and/or micro vaccine system for preventing or treating cancer based on multiple cancer cells and/or whole cell components of tumor tissue is used for preventing or treating cancer and its recurrence.
- one of the cancer cells or tumor tissues is related to the type of cancer used for prevention or treatment same.
- the loading method is the water-soluble components and water-insoluble components of the whole cells respectively or be contained inside the particle at the same time, and/or be carried on the surface of the particle separately or simultaneously.
- the whole cell components are loaded inside the nano and/or micro particles and /or the surface
- the loading method is that the water-soluble components and water-insoluble components of the whole cell are separately or simultaneously loaded inside the particle, and/or are separately or simultaneously loaded on the surface of the particle, including but not limited to water-soluble
- the ingredients are loaded in the particles and on the surface of the particles at the same time.
- the water-insoluble ingredients are loaded in the particles and on the surface of the particles at the same time.
- the water-soluble ingredients are loaded in the particles but not the water-soluble ingredients.
- the water-soluble component is loaded on the particle surface, the water-soluble component and the water-insoluble component are loaded in the particle and only the water-insoluble component is loaded on the particle surface, the water-soluble component and the water-insoluble component are loaded in the particle and only the water-soluble component is loaded.
- the ingredients are loaded on the surface of the particles, the water-soluble ingredients are loaded in the particles, the water-soluble ingredients and the water-insoluble ingredients are loaded on the particle surface at the same time, the water-insoluble ingredients are loaded in the particles, and the water-soluble ingredients and the water-insoluble ingredients are loaded on the particle surface at the same time , the water-soluble components and the water-insoluble components are simultaneously loaded in the particles and the water-soluble components and the water-insoluble components are simultaneously loaded on the surface of the particles.
- the inside and/or surface of the nano and/or micro particles also include an immune adjuvant agent.
- the method of adding the immune enhancer 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
- 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, multiple Anti-A, mineral oil, virus-like particles, immune-boosting reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin , immune adjuvant CpG, poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis
- the surface of the nano- and/or micro-vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components may not be connected to a target head with an active targeting function or may be connected to an active targeting function
- the target head; 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, basophils, one or more of lymph nodes, thymus, spleen, bone marrow.
- the whole cell components are formulated in pure water or an aqueous solution without a solubilizer
- Solubility in water can be divided into two parts: water-soluble components and non-water-soluble components.
- 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.
- the shape of the nano and/or micro particles is any common shape, including but not Limited to spheres, ellipsoids, barrels, polygons, rods, sheets, wires, worms, squares, triangles, butterflies, or discs.
- the nano and/or micro particles are particles of nanometer size and/or micron size size particles.
- the particle diameters of the nanovaccine and the nanoscale particle are respectively 1nm-1000nm, in some embodiments, the particle diameter of the nanoscale particle is 50nm-800nm, further, in some embodiments, the nanometer The particle size of the particle is 100nm-600nm.
- the particle size of the micron vaccine and the micron-sized particle is 1 ⁇ m-1000 ⁇ m, respectively.
- the particle size of the micron-sized particle is 1 ⁇ m-100 ⁇ m.
- the particle size of the micron-sized particle is The particle size is 1 ⁇ m-10 ⁇ m, further, in some embodiments, the particle size of the micron-sized particles is 1 ⁇ m-5 ⁇ m.
- the surface of the nano-sized particles or micro-sized particles can be neutrally charged, negatively charged or positively charged.
- the preparation materials of nano and/or micro particles include organic synthetic polymer materials, natural polymer materials or inorganic Material.
- the organic synthetic polymer material is a biocompatible or degradable polymer material, including but not limited to PLGA, PLA, PGA, Poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydride, PDON, PPDO , PMMA, polyamino acids, synthetic peptides, synthetic lipids.
- the natural polymer materials are biocompatible or degradable polymer materials, including but not limited to lecithin, cholesterol, starch, lipids, sugars, polypeptides, sodium alginate, albumin, collagen, gelatin, cell membrane components.
- the inorganic material is a material without obvious biological toxicity, including but not limited to ferric oxide, ferric oxide, calcium carbonate, and calcium phosphate.
- the nano and/or micro vaccine system for the prevention or treatment of cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention can deliver the loaded whole cell components to relevant immune cells, through the loading components Immunogenicity activates and enhances the killing effect of the autoimmune system on cancer cells. Therefore, the present invention also provides the application of the vaccine system for preventing or treating cancer based on various cancer cells and/or whole cell components of tumor tissues in the preparation of vaccines for preventing and/or treating cancer.
- the cancer vaccine system of the whole cell component of the present invention can simultaneously use nanoparticles and/or microparticles loaded with only water-soluble components and nanoparticles and/or microparticles loaded with only water-insoluble components when preventing or treating diseases.
- the present invention provides a nano-vaccine and/or micro-vaccine system that utilizes particles of nano-scale or micro-scale to deliver cell water-soluble components and water-insoluble components, as well as a vaccine for the prevention and treatment of cancer. application in vaccines. 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 in nanoparticles or microparticles, so most of the mutated proteins or polypeptides produced by cancer in cell components are loaded in 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 cancer vaccine system of the whole cell component of the present invention can prepare a vaccine for preventing and/or treating 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 and treating it. Cancer or prevention of cancer recurrence.
- Fig. 1 is the preparation process of vaccine described in the present invention and schematic diagram of application field; a: water-soluble component and water-insoluble component collect respectively and prepare the schematic diagram of nano-vaccine or micro-vaccine; b: adopt the solubilizing solution that contains solubilizing agent to dissolve Schematic diagram of whole cell components and preparation of nanovaccine or microvaccine.
- Figure 2- Figure 17 is a schematic diagram of the structure of nano-sized particles or micron-sized particles 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, immune enhancing adjuvant; 4, nanoparticles or microparticles; 5: the inner core part of nanoparticles; In Fig. 6-Fig. 9, the immunopotentiator is only distributed in the inside of nanoparticles or microparticles; in Fig. 10-Fig.
- the nanoparticles or microparticles only contain immune enhancing adjuvants on the outer surface; There is no immune-enhancing adjuvant inside and outside the microparticles; the water-soluble or insoluble components in the cells or tissue components loaded by nanoparticles or microparticles in Fig. 2, Fig. 6, Fig. 10 and Fig. 14 are distributed in No obvious core is formed inside the nanoparticles or microparticles; the water-soluble or non-water-soluble components in the cells or tissue components loaded by the nanoparticles or microparticles in Fig. 3, Fig. 7, Fig. 11 and Fig.
- a core part is formed inside the nanoparticle or microparticle, and the core can be generated during the preparation process or formed by using polymers or inorganic salts;
- Figure 4, Figure 8, Figure 12 and Figure 16 Nanoparticles or microparticles
- the cores can be generated during the preparation process or by using polymers or inorganic salts, etc.
- Fig. 18-Fig. 28 are schematic diagrams showing the structures of nanoparticles or microparticles loaded with water-soluble and water-insoluble cell components actively targeting the target head modification, wherein 1: water-soluble components in cell or tissue components; 2: Water-insoluble components in cells or tissue components; 3: Immunological adjuvants; 4: Nanoparticles or microparticles; 5: The core part of nanoparticles; 6: Targets that can target specific cells or tissues.
- FIG 18- Figure 19 the surface and interior of nanoparticles or microparticles contain immune adjuvants; in Figure 20-Figure 21, immune adjuvants are only distributed in the interior of nanoparticles or microparticles; Particles only contain immune adjuvant on the outer surface; Figure 24- Figure 25 has no immune adjuvant inside and outside the nanoparticle or microparticle; Figure 26 cell components and/or immune adjuvant are only distributed inside the nanoparticle or microparticle; Figure 27 The cell components and/or immune adjuvants are only distributed outside the nanoparticles or microparticles; Figure 28 The cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively.
- Figures 29-37 are respectively the nano-vaccine or micro-vaccine prepared by various cancer cells or tumor tissues in Examples 1-9 for the mouse tumor growth rate and survival experiment results when preventing or treating cancer; a, nano-vaccine or The experimental results of tumor growth rate when micron vaccines prevent or treat cancer (n ⁇ 8); b, the experimental results of mouse survival time when nano vaccines or micron vaccines prevent or treat other cancers (n ⁇ 8), each data point is 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; ** indicates that the vaccine group and Compared with the PBS blank control group, p ⁇ 0.005, there is a significant difference; ## represents the vaccine group and the blank nanoparticle + cell lysate control group, p ⁇ 0.005, there is a significant difference; *** represents the vaccine group and the PBS blank control group Compared with the control group, p ⁇ 0.0005,
- the invention discloses a broad-spectrum cancer vaccine system loaded with more than one cancer cell and/or whole cell components of tumor tissue at the nanometer or micrometer level and its application to prevent or treat cancer.
- 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 broad-spectrum cancer vaccine system loaded with more than one cancer cell and/or whole cell components of tumor tissue at the nanometer or micrometer level and its application to prevent or treat cancer.
- 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 in the aqueous solution containing a solubilizing agent.
- the solubilization solution all the cellular components can be converted into components that can be dissolved in aqueous solution and then loaded inside and outside the nanoparticles or microparticles to prepare nano-vaccine or micro-vaccine for cancer prevention and treatment .
- 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 Comprehensiveness and immunogenicity of antigenic substances or components loaded on nanoparticles or microparticles.
- 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 water-soluble part and the water-insoluble part of the cell components include the components and components of the whole cell.
- the unmutated proteins, polypeptides and genes that are the same as normal cell components will not cause immune response due to the immune tolerance generated during the development of the autoimmune system; while the mutations of genes, proteins and polypeptides produced by cancer will not cause immune responses due to the absence of an autoimmune system
- the immune tolerance developed during development is thus immunogenic and activates the body's immune response against cancer cells.
- the substances with specific immunogenicity of cancer cells produced by disease mutations in the whole cell components can be used for the prevention and treatment of cancer.
- the nano-vaccine and/or micro-vaccine system of the present invention can be used to prepare a vaccine for preventing and/or treating cancer, and its preparation process and application fields are shown in FIG. 1 .
- 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.
- 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, enzyme treatment or (and) denaturation treatment can directly prepare nano-vaccine or micro-vaccine.
- the tumor tissue cells have been inactivated or (and) denatured before lysing, and may be inactivated, enzymatically treated, or (and) denatured after cell lysing, or Inactivation, enzyme treatment or (and) denaturation treatment can also be performed before and after cell lysis; in some embodiments of the present invention, the inactivation or (and) denaturation treatment method before or (and) after cell lysis 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 actual use process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific conditions.
- the surface of the broad-spectrum cancer vaccine system of the present invention may not be connected with a target head with active targeting function or be connected with a target head with active targeting function.
- FIGS. 2-28 The structural diagrams of the vaccine system for preventing or treating cancer based on various cancer cells and/or whole cell components of tumor tissues according to the present invention are shown in FIGS. 2-28 .
- FIGS. 2-28 The structural diagrams of the vaccine system for preventing or treating cancer based on various cancer cells and/or whole cell components of tumor tissues according to the present invention are shown in FIGS. 2-28 .
- only nanoparticles and/or microparticles of a specific structure may be used, or two or more nanoparticles and/or microparticles of different structures may be used simultaneously.
- the immunopotentiator is entrapped in nanoparticles or microparticles and simultaneously loaded on the surface of nanoparticles or microparticles.
- the immunopotentiator can also be entrapped only in nanoparticles or microparticles, or It is only loaded on the surface of nanoparticles or microparticles, or no immune enhancer is added.
- the water-soluble part or (and) water-insoluble part soluble in pure water in the cell component is firstly solubilized by a solubilizer, and then encapsulated in nanoparticles or microparticles, and at the same time Immunopotentiator is loaded; then, the water-soluble part or (and) non-water-soluble part of the cell component is loaded on the surface of the nanoparticle, and the immunopotentiator is loaded at the same time. This maximizes the loading capacity of the water-soluble or water-insoluble components of the cells in the nanoparticles or microparticles.
- solubilizing solution containing a solubilizing agent such as 8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution
- a solubilizing agent such as 8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution
- the method for preparing nano-vaccine and micro-vaccine described in the present invention is a common preparation method.
- the double emulsion method in the solvent volatilization method is used to prepare the nano vaccine.
- Adjuvants are poly(I:C), Bacillus Calmette-Guerin (BCG) 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 each component in the cancer cell lysate and the immune enhancing adjuvant poly(I:C), BCG or CpG; each component in the cancer cell lysate is prepared as The water soluble component or the original water insoluble component dissolved in 8M urea.
- the concentration of the water-soluble components from cancer cells contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in the solubilizing agent from cancer cells, that is, the first predetermined concentration requires that the protein polypeptide concentration is 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 aqueous phase solution contains each component in the tumor tissue lysate and the immune enhancing adjuvant poly(I:C), BCG or CpG; each component in the tumor tissue lysate is water-soluble respectively during preparation
- the active components or the original water-insoluble components dissolved in 8M urea or the whole cell components are all dissolved in solubilizers such as 8M urea or 6M guanidine hydrochloride.
- the concentration of the water-soluble component from the tumor tissue contained in the aqueous phase solution or the concentration of the original water-insoluble component dissolved in 8M urea from the tumor tissue, that is, the first predetermined concentration requires that the protein polypeptide concentration is 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 frontal PLGA was chosen because the material is biodegradable and has been approved by the FDA for use as a drug dressing. 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.
- the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; the aqueous phase solution is a lysate component/immune adjuvant solution
- the concentration of protein and peptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL
- the concentration of immune adjuvant is greater than 0.01 ng/mL, preferably 0.01 mg/mL ⁇ 20 mg/mL.
- the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
- the concentration of the polymer material is 0.5 mg/mL ⁇ 5000 mg/mL, preferably 100 mg/mL.
- the first emulsifier solution is preferably an aqueous solution of polyvinyl alcohol, with a concentration of 10 mg/mL-50 mg/mL, preferably 20 mg/mL.
- the second emulsifier solution is preferably an aqueous polyvinyl alcohol solution with a concentration of 1 mg/mL ⁇ 20 mg/mL, preferably 5 mg/mL.
- the dispersion liquid is PBS buffer solution or physiological saline or pure water.
- Step 2 subjecting the mixed liquid obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
- the stirring is mechanical stirring or magnetic stirring
- the stirring speed is greater than 50 rpm
- the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.1 hour to 24 hours
- the ultrasonic power is greater than 5W
- the time is greater than 0.1 seconds, such as 2 to 200 seconds
- use a high-pressure/ultra-high pressure homogenizer or a high-shear homogenizer for homogenization and the pressure is greater than 5 psi when using a high-pressure/ultra-high pressure homogenizer, such as 20psi ⁇ 100psi, when using a high shear homogenizer, the speed is greater than 100 rpm, such as 1000 rpm ⁇ 5000 rpm; use microfluidics to process flow rate greater than 0.01
- Ultrasound or stirring or homogenization treatment or microfluidic treatment for nanometerization and/or micronization the length of ultrasonic time or stirring speed or homogenization treatment pressure and time can control the size of the prepared nanometer and/or micron particles, too large or too large Small will bring about changes in particle size.
- 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 homogeneous treatment or microfluidic control deal with.
- the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be nanometerized or micronized by ultrasonication or stirring.
- 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 or microparticles. Too long or too short will bring about changes in particle size. For this reason, it is necessary to choose a suitable the ultrasound 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 60-6000 seconds.
- the stirring speed is greater than 50 rpm
- the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm.
- the stirring time is 0.5 hours to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W, and the time is greater than 0.1 seconds, such as 2 to 200 seconds; Homogenizer, when using a high-pressure/ultrahigh-pressure homogenizer, the pressure is greater than 20psi, such as 20psi ⁇ 100psi, and when using a high-shear homogenizer, the speed is greater than 1000 rpm, such as 1000 rpm ⁇ 5000 rpm; use microfluidics to process flow rate greater than 0.01 mL/min, such as 0.1 mL/min-100 mL/min.
- Ultrasound or stirring or homogenization treatment or microfluidic treatment for nanometerization or micronization the length of ultrasonic time or stirring speed or homogenization treatment pressure and time can control the size of the prepared nanometer or micron particles, too large or too small will bring Changes in particle size.
- 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 may 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 stirring until predetermined stirring conditions are met.
- the emulsifier aqueous solution is still PVA.
- the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of 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 stirring condition of this step is until the organic solvent is volatilized, that is, the dichloromethane in step 1 is volatilized.
- Step 5 after centrifuging the mixed solution that meets the predetermined stirring conditions in step 4 at a speed greater than 100 rpm for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate 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 the subsequent adsorption of cancer cell lysate on the surface of nanoparticles or microparticles can be performed directly.
- 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 correlation of the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles is carried out. experiment.
- the lyoprotectant is selected from trehalose (Trehalose).
- the fifth predetermined concentration of the lyoprotectant in this step is 4% by mass, which is set so as not to 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 resuspending the nanoparticle-containing suspension obtained in step 5 with the sixth predetermined volume in PBS (or normal saline) or using the sixth predetermined volume of PBS (or normal saline) to resuspend the suspension obtained in step 6
- the freeze-dried freeze-dried substance containing nanoparticles or microparticles and a freeze-drying protective agent is mixed with the seventh predetermined volume of water-soluble components or the original water-insoluble components dissolved in 8M urea to obtain nano-vaccine or micron vaccines.
- 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 volume of the resuspended nanoparticle suspension when the volume of the resuspended nanoparticle suspension is 10 mL, it contains cancer cell lysates or contains water-soluble components in tumor tissue lysates or original water-insoluble components dissolved in 8M urea.
- the volume of the components is 1 mL. The volume and ratio of the two can be adjusted as required during actual use.
- the used water-soluble components containing cancer cell lysates or tumor tissue lysates or the original non-water-soluble components dissolved in 8M urea contain poly(I:C), Bacillus Calmette-Guerin (BCG) or CpG, and the concentration of poly(I:C), BCG or CpG is greater than 1 ng/mL.
- the particle size of the nano-vaccine or micro-vaccine is nanometer or micrometer, which can ensure that the vaccine is phagocytized by antigen-presenting cells, and 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 cancer cell lysates or tumor tissue lysates, and any other components that can make cancer cell lysates or tumor tissue lysates
- 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
- 8M urea and 6M guanidine hydrochloride aqueous solution are used to solubilize the original water-insoluble components in cancer cell lysates or tumor tissue lysates, and any other components that can make cancer cells
- the original water-insoluble components in the cell lysate or tumor tissue lysate are dissolved in the urea concentration or the guanidine hydrochloride concentration of the aqueous solution; or the water-soluble components and the water-insoluble components are simultaneously dissolved using 8M urea aqueous solution.
- 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 lysate or the tumor tissue lysate or the original water-insoluble components dissolved in 8M urea are respectively loaded inside the nanoparticles and adsorbed on the surface of the nanoparticles.
- the water-soluble components in the cancer cell lysate or tumor tissue lysate and the original water-insoluble components dissolved in 8M urea can also be mixed and then loaded into the interior of the nanoparticles or loaded onto the surface of the nanoparticles; or It is also possible to use 8M urea to simultaneously dissolve the water-soluble component and the water-insoluble component and then entrap the inside of the nanoparticle or microparticle and/or adsorb on the surface of the nanoparticle or microparticle.
- Loading methods include chemical bonding and/or non-chemical bonding adsorption.
- poly(I:C), manganese adjuvant, Bacillus Calmette-Guerin (BCG) and CpG are used as immune adjuvants.
- BCG Bacillus Calmette-Guerin
- CpG CpG
- no immune adjuvant or any other immune adjuvant with immune enhancing function can be added.
- Agents such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyclonal A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virus Body, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant poly ICLC, Corynebacterium pumilus vaccine, hemolytic streptococcus preparation , coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum adjuvant, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM - CSF, MF59, double-stranded RNA, double-stranded DNA
- 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 and/or micro-vaccine according to the actual situation.
- 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 nano-sized particle or micron-sized particle structure, preparation method, and use strategy during disease treatment mentioned in the embodiments of the present invention are only representative methods, and other nano-sized particles or micron-sized particle structures, preparation methods, disease prevention or The use strategy during treatment 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.
- Example 1 The whole cell components of melanoma tumor tissue and lung cancer tumor tissue are loaded inside and on the surface of nanoparticles for the prevention of melanoma: this example uses mouse melanoma as a cancer model to illustrate how to prepare melanoma tumor tissue and whole-cell components of lung cancer tumor tissue as a nanovaccine, and apply the vaccine to prevent melanoma.
- B16F10 mouse melanoma cells were used as the cancer model. Firstly, the B16F10 melanoma tumor tissue and the LLC lung cancer tumor tissue were lysed to prepare the water-soluble fraction and the water-insoluble fraction of the 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 the water-soluble component and the water-insoluble component loaded with tumor tissue by the solvent evaporation method. Divided nano-vaccine. The nanovaccine was then employed to prevent melanoma.
- the tissue cells After the tissue cells are lysed, centrifuge the lysate at a speed of 5000g for 3 minutes and take the supernatant, which is the water-soluble component soluble in pure water; The water-insoluble components of pure water were converted to be soluble in 8M aqueous urea solution.
- the water-soluble components from B16-F10 tumor tissue and LLC lung cancer tumor tissue and the original water-insoluble components dissolved in 8M urea were mixed in a ratio of 1:1, which was the source of raw materials for preparing vaccines.
- this embodiment adopts water-soluble polypeptide B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM) in equal proportions, B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M27 (REGVELCPGNKYEMRRHGTTHSLVIHD) and 8M urea solubilized water-insoluble polypeptide B16 -M05 (Eef2, FVVKAYLPVNESFAFTADLRSNTGGQA), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ), and TRP2:180-188 (SVYDFFVWL).
- nano-vaccine In this example, the nano-vaccine, the blank nano-particles used as the control, and the nanoparticles loaded with various polypeptides were prepared by the double emulsion method in the solvent evaporation method, and the molecular weight of the nano-particle preparation material PLGA used was 24KDa-38KDa, the immune adjuvant used is poly(I:C) And poly(I:C) is not only distributed inside the nanoparticles but also loaded on the surface of the nanoparticles.
- the preparation method is as described above.
- the average particle size of the nano-vaccine loaded with whole cell components is about 320nm, and the surface potential of the nano-vaccine is about-5mV; every 1mg PLGA nano-particle is about loaded with 180 ⁇ g protein or polypeptide component, and the poly(I: C)
- the immune adjuvant is about 0.01 mg in total and half inside and outside.
- the particle size of blank nanoparticles is about 270nm.
- the preparation method of polypeptide-loaded nanoparticles is the same as the preparation method of preparing whole-cell components, and the amount of poly(I:C) used is also the same.
- the particle size of polypeptide nanoparticles is about 310nm, and every 1mg PLGA nanoparticles are loaded with about 150 ⁇ g of polypeptide components.
- the water-soluble component from B16-F10 tumor tissue and the original water-insoluble component dissolved in 8M urea were used as raw materials to prepare the vaccine loaded only with the whole cell component of B16F10 tumor tissue.
- Nano-vaccine for the prevention of cancer the control groups in this study were the PBS group and the blank nanoparticle+free tissue lysate group. Select 6-8-week-old female C57BL/6 as model mice to prepare melanoma-bearing mice.
- the administration regimen of the nanovaccine group was as follows: 200 ⁇ L of 2 mg PLGA nanovaccine loaded with water-soluble components on the inside and on the surface were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation and 200 ⁇ L of 2 mg PLGA nano-vaccine loaded with original water-insoluble components on the inside and on the surface; on day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse.
- the protocol of the PBS control group is as follows: 400 ⁇ L PBS was injected subcutaneously on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation; on the 0th day, 1.5 ⁇ 105 mice were subcutaneously inoculated on the lower right back of each mouse B16F10 cells.
- Blank nanoparticles + free lysate control group 400 ⁇ L of blank nanoparticles and the same amount of free lysate loaded with the vaccine were subcutaneously injected on days 49, 42, 35, 28 and 14 before inoculation of melanoma; blank 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.
- the dosage regimen of the polypeptide nanoparticle group was as follows: 200 ⁇ L of 2 mg PLGA nanovaccine loaded with water-soluble components on the inside and on the surface were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. and 200 ⁇ L of 2 mg PLGA nano-vaccine loaded with the original water-insoluble components both inside and on the surface; on day 0, 1.5 ⁇ 10 5 B16F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse.
- v was the tumor volume
- a was the tumor length
- b was the tumor width.
- Example 2 Water-soluble cell components of melanoma and lung cancer cells are loaded on the inside and surface of microparticles for the prevention of melanoma:
- This example uses mouse melanoma as a cancer model to illustrate how to prepare only melanoma and lung cancer cells A micron vaccine of the water-soluble part in the composition, and apply the vaccine to prevent melanoma.
- B16F10 melanoma and LLC lung cancer cells were firstly lysed to prepare water-soluble fractions and water-insoluble fractions. Then, the organic polymer material PLGA (38KDa-54KDa) was used as the micron particle skeleton material, and poly(I:C) was used as the immune adjuvant to prepare the micron vaccine loaded with the whole cell water-soluble component by the solvent evaporation method. The micron vaccine was then used to prevent melanoma.
- Lysis of cancer cells and collection of various components collect a certain amount of B16F10 cells or LLC cells, remove the culture medium and freeze at -20°C, add a certain amount of ultrapure water and freeze-thaw more than 3 times repeatedly. Accompanied by sonication to disrupt lysed cells. 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 soluble in pure water in B16F10 melanoma or LLC lung cancer cells. The above-mentioned water-soluble components derived from two kinds of cancer cell lysates are mixed at a ratio of 1:1, which is the antigen source for preparing micron vaccines.
- 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 volatilization method, the micron particle preparation material used is PLGA, and the immune adjuvant used is CPG And the CPG is not only distributed inside the microparticles but also loaded on the surface of the microparticles.
- the preparation method is as described above.
- the particle size of the micron vaccine obtained after loading cell components and immune adjuvant on the surface of the micron particle is about 1.40 ⁇ m, and the average surface potential of the micron particle is about -5mV.
- Each 1 mg of PLGA microparticles is loaded with 210 ⁇ 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, with half and half inside.
- the particle size of the blank microparticles is about 1.20 ⁇ 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 scheme of the Micron vaccine group was as follows: 400 ⁇ L of 4 mg PLGA Micron vaccine loaded with water-soluble components in cancer cell lysates were subcutaneously injected on the 28th day, 21st day, and 14th day before the melanoma inoculation; 1.5 ⁇ 105 B16F10 cells were inoculated subcutaneously in the lower right lower back of each mouse.
- the protocol for the PBS blank control group was as follows: 400 ⁇ L PBS was subcutaneously injected on the 28th day, 21st day, and 14th day before the inoculation of melanoma; on the 0th day, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse. cell.
- 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.
- 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
- Example 3 Lung cancer tumor tissue and liver cancer tumor tissue lysate components are loaded inside and on the surface of microparticles for the prevention of liver cancer: This example is how to prepare micron vaccines loaded with liver cancer and lung cancer tumor tissue lysate components, and apply this Vaccines Prevent Liver Cancer to illustrate how vaccines prepared from tumor tissue can be used to prevent liver cancer.
- mouse liver cancer and lung cancer tumor tissue lysates were loaded on the interior and surface of nanoparticles at a ratio of 1:3 to prepare micron vaccines.
- the mouse lung cancer and liver cancer tumor tissues were obtained and lysed to prepare the water-soluble fraction of the tumor mass tissue and the original water-insoluble fraction dissolved in 8M urea.
- PLGA 24KDa-38KDa
- poly(I:C) is used as the immune adjuvant to prepare the micron vaccine.
- the micron vaccine is then used to prevent Hepa 1-6 Tumors in HCC tumor-bearing mice.
- Micron vaccine for cancer prevention select 6-8 week old female C57BL/6 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 micron vaccine loaded with water-soluble components on the inside and on the surface and 200 ⁇ L of original non- 2mg PLGA micron vaccine of water soluble composition.
- 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.
- Example 4 Whole cell components of lung cancer and liver cancer tumor tissues are loaded inside nanoparticles for the prevention of liver cancer:
- This example uses mouse liver cancer as a cancer model to illustrate how to prepare nanoparticles loaded with whole cell components of lung cancer and liver cancer tumor tissues. vaccine, and use the vaccine to prevent liver cancer.
- Lung cancer and liver cancer tumor tissues were first lysed to prepare water-soluble and water-insoluble fractions of the whole cell fraction and mixed at a ratio of 1:2. Then, using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant, the nanovaccine loaded with water-soluble components and water-insoluble components of lung cancer tumor mass and liver cancer tumor mass was prepared by solvent evaporation method. This nanovaccine was then employed to prevent liver cancer.
- the preparation of nano-vaccine adopts the double emulsion method in the solvent evaporation method
- the nanoparticle preparation material PLGA molecular weight used is 24KDa-38KDa
- the immune adjuvant used is poly(I:C) and poly(I:C) are both distributed inside the nanoparticles and adsorbed on the surface of the nanoparticles.
- the water-soluble component is a mixture of the water-soluble components of the lung cancer tumor tissue and the water-soluble components of the liver cancer tumor tissue, which are only distributed inside the vaccine; the water-insoluble components are the non-water-soluble components of the lung cancer tumor tissue and the A mixture of water-insoluble components of tumor tissue, distributed only inside the vaccine.
- the particle size of the nano-vaccine obtained after adsorption of the immune adjuvant on the surface of the nanoparticles is about 300nm, and the average surface potential Zeta potential of the nanoparticles 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, and the inside and outside are divided in half.
- the particle size of blank nanoparticles is about 230nm. Pure water containing poly (I:C) or 8M urea were used to replace the corresponding water-soluble components and insoluble components when preparing blank nanoparticles. The surface adsorption of blank nanoparticles and Equivalent poly(I:C) in nanovaccine.
- Nano-vaccine for cancer prevention the administration scheme and tumor growth monitoring scheme of the vaccine group and the PBS control group are as in Example 3.
- Example 5 Pancreatic cancer tumor tissue and colon cancer tumor tissue lysed components 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 pancreatic cancer tumor tissue and colon cancer tumor tissue lysate components, and apply the vaccine to treat pancreatic cancer.
- mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue lysate fractions were loaded on the inside and surface of nanoparticles at a mass ratio of 2:1 to prepare a nanovaccine.
- mouse pancreatic and colon cancer tumor tissues were obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea.
- the water-soluble component is a 2:1 mixture of the water-soluble component of pancreatic cancer tumor tissue and the water-soluble component of colon cancer tumor tissue; the water-insoluble component is the water-insoluble component of pancreatic cancer tumor tissue and the A 2:1 mixture of water-insoluble components of cancerous tumor tissue.
- the nanovaccine was prepared by the solvent evaporation method. The nanovaccine was then used to treat tumors in Pan02 pancreatic cancer-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 400 ⁇ L of 4 mg PLGA nanoparticles loaded with water-soluble components inside and loaded with original water-insoluble components on the surface on the 4th day, 7th day, 10th day, 15th day and 20th day respectively.
- 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.
- mouse tumor monitoring and volume calculation methods were the same as above.
- Example 6 Whole cell components of breast cancer and lung cancer tumor tissues loaded inside mannose-modified microparticles for the prevention of lung cancer:
- This example uses mouse lung cancer as a cancer model to illustrate how to prepare breast cancer and lung cancer tumor tissues loaded A micron vaccine of whole cell components, and using 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.
- the lysed components of mouse breast cancer and lung cancer tumor tissues were loaded inside the microparticles at a ratio of 1:4 to prepare micron vaccines.
- Tumor tissues of mouse breast and lung cancers were first obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea. Then, using PLGA and mannose-modified PLGA as the microparticle framework material, and CpG as the immune adjuvant, the microvaccine was prepared by the solvent evaporation method.
- the micron vaccine has the ability to target dendritic cells.
- micro-vaccine Preparation of micro-vaccine:
- the micro-vaccine and the empty micro-particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micro-particle preparation material PLGA (50:50) is used
- the molecular weight is 38KDa-54KDa
- the molecular weight of the mannose-modified PLGA (50:50) used is 38KDa-54KDa.
- the mass ratio of unmodified PLGA to mannose-modified PLGA is 8:2.
- the water-soluble components in the cancer cell lysate are loaded inside the micron vaccine, and the non-water-soluble components dissolved in 8M urea are loaded on the surface of the vaccine.
- the immune adjuvant used is CpG and CpG is distributed inside the micron particles.
- the preparation method is as mentioned above.
- the average particle size of the micron particles is about 1.30 ⁇ m, and the average surface potential is about -9 mV. per 1 mg PLGA microparticles are loaded with 65 ⁇ g of protein or polypeptide components, and the CpG immune adjuvant used inside and outside of each 1 mg of PLGA microparticles is 0.025 mg.
- the particle size of the blank microparticles is about 1.20 ⁇ 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 cancer prevention 6-8 week old female C57BL/6 were selected as model mice to prepare lung cancer tumor-bearing mice.
- the vaccine group was subcutaneously injected with 400 ⁇ L of 4 mg PLGA micron vaccine on days 35, 28, 21, 14 and 7 before tumor inoculation.
- 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 + lysate control group were subcutaneously injected with 400 ⁇ L of blank microparticles 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 ⁇ 10 6 LLC lung cancer cells were subcutaneously inoculated into the lower right lower back of each mouse. In the experiment, the mouse tumor growth monitoring method was the same as above.
- Example 7 The whole cell components of liver cancer and melanoma tumor tissue are loaded on the inside and surface of nanoparticles and the nano-vaccine with Bacillus Calmette-Guerin (BCG) as an immune adjuvant is used for the prevention of liver cancer:
- BCG Bacillus Calmette-Guerin
- mouse liver cancer was used as a cancer model and Using BCG as an immune adjuvant to illustrate how to prepare nano-vaccine loaded with whole tissue cell components of melanoma and liver cancer tumors and apply the vaccine to prevent liver cancer.
- the water-soluble and water-insoluble components of liver cancer and melanoma tumor tissues were firstly lysed and mixed at a ratio of 3:1. Then, using PLGA as the nanoparticle framework material and BCG as the immune adjuvant, the nanovaccine was prepared by the solvent evaporation method.
- Lysis of tumor tissue and collection of various components Lysis of tumor tissue and collection of lysate in this embodiment are the same as above.
- Nano-vaccine for the prevention of liver cancer select female C57BL/6 as model mice to prepare liver cancer tumor-bearing mice.
- the vaccine group received subcutaneous injections of 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in cancer cell lysate and 200 ⁇ L of 2 mg PLGA nanoparticles dissolved in 8M urea, the original water-insoluble components, were loaded on the surface.
- 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 ⁇ 10 6 Hepa1-6 liver cancer cells were subcutaneously inoculated into the lower right back of each mouse.
- the mouse tumor growth monitoring method was the same as above.
- Example 8 6M guanidine hydrochloride dissolves tumor tissue components of breast cancer and colon cancer and loads them inside and on the surface of microparticles for the treatment of breast cancer: This example illustrates how to use 6M guanidine hydrochloride to dissolve whole cell components and prepare whole cell components loaded with microparticles Micro-vaccines of cellular components to treat breast cancer.
- 4T1 mouse triple-negative breast cancer was used as a cancer model. First, inactivate and denature the tumor tissue cells of breast cancer and colon cancer, lyse the tumor tissue with 6M guanidine hydrochloride and dissolve the whole cell components.
- the micron vaccine loaded with the whole cell components of the tumor tissue was prepared by the solvent evaporation method.
- the microvaccine was then used to treat tumors in 4T1 breast cancer tumor-bearing mice.
- the obtained tumor tissue cells were inactivated and denatured respectively by ultraviolet light and high temperature heating, and then 6M guanidine hydrochloride was used to lyse the breast cancer and colon cancer tumor tissue cells and dissolve the tissue lysates, and the breast cancer tumor tissue lysates were combined with the colon cancer tumor tissue
- the lysate is mixed according to the ratio of 5:1, which is the raw material source for preparing the vaccine.
- micron vaccine Preparation of micron vaccine:
- 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 2.6 ⁇ m, and the Zeta potential on the surface 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 0.02 mg in total, and the inside and outside are divided into half.
- Unadjuvanted micron vaccines have the same properties as CpG-adjuvanted vaccines, except that they do not contain CpG adjuvant.
- 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.
- the control group of blank microparticles + free lysate was subcutaneously injected with the same 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 lysate 4mg PLGA blank micron particles of the material composition.
- the mouse tumor volume was monitored and calculated in the same way as above.
- Example 9 Whole cell components of melanoma tumor tissue and lung cancer cancer cells loaded on the inside and surface of nanoparticles for the prevention of melanoma: This example illustrates how to prepare whole cell components loaded with melanoma tumor tissue and lung cancer cancer cells nano-vaccine and apply the vaccine to prevent melanoma.
- B16F10 melanoma tumor tissue and LLC cancer cells were lysed to prepare corresponding water-soluble fractions and water-insoluble fractions dissolved in 8M urea.
- the water-soluble components from tumor tissue and the water-soluble components from cancer cells were mixed at a mass ratio of 1:1 to be the water-soluble components used in the experiment; the water-insoluble components from tumor tissues and the water-soluble components from cancer cells
- the water-insoluble components of the cells are mixed according to the mass ratio of 1:1, which is the water-insoluble components used in the experiment; then, the nano-vaccine is prepared with PLGA as the skeleton material and colloidal manganese as the immune adjuvant.
- 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 allowed to react at room temperature for 10 min before use.
- the average particle size of the nanovaccine loaded with whole cell components is about 520nm, and the surface potential of the nanovaccine is about -5mV; each 1 mg PLGA nanoparticle is loaded with about 190 ⁇ g protein or polypeptide component.
- the particle size of the blank nanoparticles is about 470nm, and the blank nanoparticles are loaded with the same 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.
Abstract
A vaccine system for preventing or treating cancer on the basis of multiple cancer cells and/or tumor tissue whole cell components, and a preparation method therefor and an application thereof. A water-soluble component and a non-water-soluble component of the whole cell components mixed with a plurality of cells are delivered by using nano-scale or micron-sized particles for preparing a vaccine for preventing and treating cancer. Both the water-soluble portion and the non-water-soluble portion are loaded in a nano-vaccine or a micro-vaccine, so that a variant protein or polypeptide generated by cancer in the cell components is loaded in the nano-vaccine or the micro-vaccine. Among the whole cell components, these substances, which have immunogenicity and are produced due to illness and mutation, can be used for preventing and treating cancer. Therefore, the nano-vaccine and/or micro-vaccine system of the whole cell components of different cell sources can be used for preparing a drug for preventing and/or treating cancer.
Description
本发明属于免疫治疗领域，具体涉及一种基于不同癌细胞和/或不同肿瘤组织的纳米或微米的癌症疫苗，尤其是涉及一种基于两种及以上癌症的癌细胞和/或肿瘤组织的全细胞组分的癌症疫苗及其在预防和治疗癌症中的应用。The invention belongs to the field of immunotherapy, in particular to a nano or micro cancer vaccine based on different cancer cells and/or different tumor tissues, in particular to a whole-body vaccine based on two or more cancer cells and/or tumor tissues Cancer vaccines of cellular components and their use in the prevention and treatment of cancer.
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，从而破坏和清除人体内的异常物质（如病毒、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。近些年来免疫技术发展很快，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 removing abnormal substances (such as viruses, bacteria, etc.) in the human body, or damaged cells produced by the human body itself and tumor cells to maintain human health. Immunotechnology has developed 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 balance of the body's immune system, it is expected to affect and control the occurrence, development and treatment of cancer.
癌症疫苗在癌症免疫治疗和预防的重要方法之一。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的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。现有技术都是采用一种癌细胞或者肿瘤组织全细胞构建癌症疫苗系统，用于该癌症的治疗，还未见采用多种癌症细胞或肿瘤组织制备的癌症疫苗用于更好的预防和治疗癌症的报道。Cancer vaccines are one of the important approaches in cancer immunotherapy and prevention. 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 whole cell components of cancer cells or cancer tissues as a source of vaccines for the prevention and treatment of cancer. In the prior art, a cancer vaccine system is constructed by whole cells of a cancer cell or tumor tissue for the treatment of the cancer. There is no cancer vaccine prepared by using a variety of cancer cells or tumor tissue for better prevention and treatment. Cancer Reports.
有鉴于此，本发明的目的在于针对现有技术存在的问题，提供一种负载多种癌症细胞和/或肿瘤组织的全细胞组分的微米或纳米疫苗系统用于预防或治疗癌症的方法。In view of this, the object of the present invention is to solve the problems existing in the prior art, and provide a method for preventing or treating cancer with a micro or nano vaccine system loaded with whole cell components of various cancer cells and/or tumor tissues.
为实现本发明的目的，本发明采用如下技术方案：一种基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，包括纳米和/或微米粒子、全细胞组分混合物；所述全细胞组分混合物为多种癌细胞和/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 scheme: a nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components, including nano and/or micro Particles, a mixture of whole cell components; the mixture of whole cell components is whole cell components of various cancer cells and/or tumor tissues.
本发明基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的疫苗系统为纳米或微米的疫苗系统，称为纳米疫苗或微米疫苗，可以预防或治疗癌症，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混合物组成，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混合物、免疫佐剂组成；所述全细胞组分为不同癌细胞和/或不同肿瘤组织中全细胞的水溶性成分混合物和/或非水溶性成分混合物。混合物可为但不限于水溶性成分互相混合，或者非水溶性成分互相混合，或者全部和/或部分水溶性组分与全部和/或部分水溶性组分互相混合。The vaccine system for the prevention or treatment of cancer based on a variety of cancer cells and/or tumor tissue whole cell components is a nano or micro vaccine system, called a nano vaccine or a micro vaccine, which can prevent or treat cancer. or a mixture of micron-sized particles and said particle-loaded whole cell components, or a mixture of nano-sized or micron-sized particles and said particle-loaded whole cell components, and an immune adjuvant; said whole The cell component is a mixture of water-soluble components and/or a mixture of water-insoluble components of whole cells in different cancer cells and/or different tumor tissues. The mixture can be, but is not limited to, water soluble ingredients mixed with each other, or water insoluble ingredients mixed with each other, or all and/or partially water soluble components mixed with all and/or partially water soluble components.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统的制备方法为，将多种癌细胞和/或肿瘤组织全细胞组分负载于纳米和/或微米粒子内部和/或表面，得到所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统；或者将多种癌细胞和/或肿瘤组织全细胞组分、免疫佐剂负载于纳米和/或微米粒子内部和/或表面，得到所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统。具体的，本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的疫苗系统可以按照纳米尺寸粒子和微米尺寸粒子已开发的制备方法制备得到，包括但不仅限于常见的溶剂挥发法、透析法、挤出法、热熔法。在一些实施方案中，所述的疫苗系统采用溶剂挥发法中的复乳法制备得到。The preparation method of the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention is: Loaded inside and/or on the surface of nano and/or micro particles to obtain the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components; Cells and/or tumor tissue whole cell components and immune adjuvants are loaded inside and/or on the surface of nanometer and/or micron particles to obtain the prevention or treatment of cancer based on a variety of cancer cells and/or tumor tissue whole cell components nano and/or micro vaccine systems. Specifically, the vaccine system for preventing or treating cancer based on a variety of cancer cells and/or tumor tissue whole cell components of the present invention can be prepared according to the preparation methods developed for nano-sized particles and micron-sized particles, including but not limited to Common solvent evaporation method, dialysis method, extrusion method, hot melt method. In some embodiments, the vaccine system is prepared by the double emulsion method in the solvent evaporation method.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统的活性成分全细胞组分混合物为全细胞的水溶性成分混合物和/或非水溶性成分混合物，由两种或两种以上的癌细胞和/或肿瘤组织制备，可以为两种或两种以上的癌细胞制备，也可以由两种或两种以上的肿瘤组织制备，亦可以由一种以上的癌细胞和肿瘤组织制备。此为本发明的创造性所在，现有技术都是采用一种癌细胞或者肿瘤组织制备全细胞组分，用于纳米和/或微米粒子构建癌症疫苗，本发明采用两种或以上癌细胞和/或肿瘤组织制备癌症疫苗，预料不到的取得技术效果显著进步。The active ingredient whole cell component mixture of the nanometer and/or micron vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention is a water-soluble component mixture of whole cells and/or A mixture of water-insoluble components, prepared from two or more cancer cells and/or tumor tissues, can be prepared from two or more cancer cells, or can be prepared from two or more tumor tissues, It can also be prepared from more than one kind of cancer cells and tumor tissues. This is where the creativity of the present invention lies. In the prior art, one kind of cancer cell or tumor tissue is used to prepare whole cell components for nano and/or micro particle construction of cancer vaccines. The present invention uses two or more cancer cells and/or Or tumor tissue to prepare cancer vaccines, unexpectedly achieved significant progress in technical effects.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统用于预防或治疗癌症及其复发。本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，癌细胞或肿瘤组织中有一种与用于预防或治疗的癌症类型相同。The nano and/or micro vaccine system for preventing or treating cancer based on multiple cancer cells and/or whole cell components of tumor tissue is used for preventing or treating cancer and its recurrence. In the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, one of the cancer cells or tumor tissues is related to the type of cancer used for prevention or treatment same.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，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。In the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the loading method is the water-soluble components and water-insoluble components of the whole cells respectively or be contained inside the particle at the same time, and/or be carried on the surface of the particle separately or simultaneously.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，所述全细胞组分被负载于纳米和/或微米粒子内部和/或表面，具体的，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，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 nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the whole cell components are loaded inside the nano and/or micro particles and /or the surface, specifically, the loading method is that the water-soluble components and water-insoluble components of the whole cell are separately or simultaneously loaded inside the particle, and/or are separately or simultaneously loaded on the surface of the particle, including but not limited to water-soluble The ingredients are loaded in the particles and on the surface of the particles at the same time. The water-insoluble ingredients are loaded in the particles and on the surface of the particles at the same time. The water-soluble ingredients are loaded in the particles but not the water-soluble ingredients. In the particle, the water-soluble component is loaded on the particle surface, the water-soluble component and the water-insoluble component are loaded in the particle and only the water-insoluble component is loaded on the particle surface, the water-soluble component and the water-insoluble component are loaded in the particle and only the water-soluble component is loaded The ingredients are loaded on the surface of the particles, the water-soluble ingredients are loaded in the particles, the water-soluble ingredients and the water-insoluble ingredients are loaded on the particle surface at the same time, the water-insoluble ingredients are loaded in the particles, and the water-soluble ingredients and the water-insoluble ingredients are loaded on the particle surface at the same time , the water-soluble components and the water-insoluble components are simultaneously loaded in the particles and the water-soluble components and the water-insoluble components are simultaneously loaded on the surface of the particles.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，所述纳米和/或微米粒子内部和/或表面还包括免疫佐剂。所述的免疫增强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、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、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 nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the inside and/or surface of the nano and/or micro particles also include an immune adjuvant agent. The method of adding the immune enhancer 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 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, multiple Anti-A, mineral oil, virus-like particles, immune-boosting reconstituted influenza virions, cholera enterotoxin, saponins and their derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharides, curcumin , immune adjuvant CpG, poly(I:C), immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, poly muscle nucleotide, polyadenylic acid, alum, 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 astragalus. Those skilled in the art can understand that the immune enhancing adjuvant can also use other substances that can enhance the immune response.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米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。The surface of the nano- and/or micro-vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components may not be connected to a target head with an active targeting function or may be connected to an active targeting function The target head; 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, basophils, one or more of lymph nodes, thymus, spleen, bone marrow.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，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 nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the whole cell components are formulated in pure water or an aqueous solution without a solubilizer Solubility in water can be divided into two parts: water-soluble components and non-water-soluble components. 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.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，纳米和/或微米粒子的形状为常见的任意形状，包括但不限于球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形。本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统中，纳米和/或微米粒子为纳米级尺寸的粒子和/或微米级尺寸的粒子。所述纳米疫苗和纳米级尺寸的粒子的粒径分别为1nm-1000nm，在一些实施方案中，所述纳米尺寸粒子的粒径为50nm-800nm，进一步的，在一些实施方案中，所述纳米尺寸粒子的粒径为100nm-600nm。所述微米疫苗和微米级尺寸的粒子的粒径分别为1μm-1000μm，在一些实施方案中，所述微米尺寸粒子的粒径为1μm-100μm，在一些实施方案中，所述微米尺寸粒子的粒径为1μm-10μm，进一步的，在一些实施方案中，所述微米尺寸粒子的粒径为1μm-5μm。所述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 nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the shape of the nano and/or micro particles is any common shape, including but not Limited to spheres, ellipsoids, barrels, polygons, rods, sheets, wires, worms, squares, triangles, butterflies, or discs. In the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the nano and/or micro particles are particles of nanometer size and/or micron size size particles. The particle diameters of the nanovaccine and the nanoscale particle are respectively 1nm-1000nm, in some embodiments, the particle diameter of the nanoscale particle is 50nm-800nm, further, in some embodiments, the nanometer The particle size of the particle is 100nm-600nm. The particle size of the micron vaccine and the micron-sized particle is 1 μm-1000 μm, respectively. In some embodiments, the particle size of the micron-sized particle is 1 μm-100 μm. In some embodiments, the particle size of the micron-sized particle is The particle size is 1 μm-10 μm, further, in some embodiments, the particle size of the micron-sized particles is 1 μm-5 μm. The surface of the nano-sized particles or micro-sized particles can be neutrally charged, negatively charged or positively charged.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的疫苗系统中，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。其中所述有机合成高分子材料为生物相容或可降解的高分子材料，包括但不限于PLGA、PLA、PGA、Poloxamer、PEG、PCL、PEI、PVA、PVP、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽、合成脂质。所述的天然高分子材料为生物相容或可降解的高分子材料，包括但不限于卵磷脂、胆固醇、淀粉、脂类、糖类、多肽、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜成分。所述的无机材料为无明显生物毒性的材料，包括但不限于三氧化二铁、四氧化三铁、碳酸钙、磷酸钙。In the vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the preparation materials of nano and/or micro particles include organic synthetic polymer materials, natural polymer materials or inorganic Material. Wherein the organic synthetic polymer material is a biocompatible or degradable polymer material, including but not limited to PLGA, PLA, PGA, Poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydride, PDON, PPDO , PMMA, polyamino acids, synthetic peptides, synthetic lipids. The natural polymer materials are biocompatible or degradable polymer materials, including but not limited to lecithin, cholesterol, starch, lipids, sugars, polypeptides, sodium alginate, albumin, collagen, gelatin, cell membrane components. The inorganic material is a material without obvious biological toxicity, including but not limited to ferric oxide, ferric oxide, calcium carbonate, and calcium phosphate.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统可将装载的全细胞组分递送给相关免疫细胞，通过所装载成分的免疫原性而激活和增强自身免疫系统对癌细胞的杀伤作用。因此本发明还提供了所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的疫苗系统在制备预防和/或治疗癌症的疫苗中的应用。The nano and/or micro vaccine system for the prevention or treatment of cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention can deliver the loaded whole cell components to relevant immune cells, through the loading components Immunogenicity activates and enhances the killing effect of the autoimmune system on cancer cells. Therefore, the present invention also provides the application of the vaccine system for preventing or treating cancer based on various cancer cells and/or whole cell components of tumor tissues in the preparation of vaccines for preventing and/or treating cancer.
本发明所述全细胞组分的癌症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和/或微米粒子和只负载非水溶性组分的纳米粒子和/或微米粒子、使用只负载水溶性组分的纳米粒子和/或微米粒子、使用只负载非水溶性组分的纳米粒子和/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和/或微米粒子。The cancer vaccine system of the whole cell component of the present invention can simultaneously use nanoparticles and/or microparticles loaded with only water-soluble components and nanoparticles and/or microparticles loaded with only water-insoluble components when preventing or treating diseases. Particles, using nanoparticles and/or microparticles loaded with only water-soluble components, using nanoparticles and/or microparticles loaded only with water-insoluble components, or using nanoparticles loaded with both water-soluble components and water-insoluble components particles and/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非水溶性成分的纳米疫苗和/或微米疫苗系统，以及用于制备预防和治疗癌症的疫苗中的应用。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中，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中。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。It can be seen from the above technical scheme that the present invention provides a nano-vaccine and/or micro-vaccine system that utilizes particles of nano-scale or micro-scale to deliver cell water-soluble components and water-insoluble components, as well as a vaccine for the prevention and treatment of cancer. application in vaccines. 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 in nanoparticles or microparticles, so most of the mutated proteins or polypeptides produced by cancer in cell components are loaded in 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.
本发明所述全细胞组分的癌症疫苗系统可以制备预防和/或治疗癌症的疫苗。在用作癌症疫苗以预防和治疗癌症时，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 cancer vaccine system of the whole cell component of the present invention can prepare a vaccine for preventing and/or treating cancer. When used as a cancer vaccine to prevent and treat 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 and treating it. Cancer or prevention of cancer recurrence.
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为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. 1 is the preparation process of vaccine described in the present invention and schematic diagram of application field; a: water-soluble component and water-insoluble component collect respectively and prepare the schematic diagram of nano-vaccine or micro-vaccine; b: adopt the solubilizing solution that contains solubilizing agent to dissolve Schematic diagram of whole cell components and preparation of nanovaccine or microvaccine.
图2-图17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粒子的结构示意图，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；图2-图5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6-图9中免疫增强剂只分布于纳米粒子或微米粒子的内部；图10-图13中纳米粒子或微米粒子只在外表面含有免疫增强佐剂；图14-图17纳米粒子或微米粒子内部和外表面均无免疫增强佐剂；图2，图6，图10和图14中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3，图7，图11和图15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4，图8，图12和图16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5，图9，图13和图17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。Figure 2-Figure 17 is a schematic diagram of the structure of nano-sized particles or micron-sized particles 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, immune enhancing adjuvant; 4, nanoparticles or microparticles; 5: the inner core part of nanoparticles; In Fig. 6-Fig. 9, the immunopotentiator is only distributed in the inside of nanoparticles or microparticles; in Fig. 10-Fig. 13, the nanoparticles or microparticles only contain immune enhancing adjuvants on the outer surface; There is no immune-enhancing adjuvant inside and outside the microparticles; the water-soluble or insoluble components in the cells or tissue components loaded by nanoparticles or microparticles in Fig. 2, Fig. 6, Fig. 10 and Fig. 14 are distributed in No obvious core is formed inside the nanoparticles or microparticles; the water-soluble or non-water-soluble components in the cells or tissue components loaded by the nanoparticles or microparticles in Fig. 3, Fig. 7, Fig. 11 and Fig. 15 are distributed in A core part is formed inside the nanoparticle or microparticle, and the core can be generated during the preparation process or formed by using polymers or inorganic salts; Figure 4, Figure 8, Figure 12 and Figure 16 Nanoparticles or microparticles When the water-soluble or non-water-soluble components in the loaded cells or tissue components are distributed inside the nanoparticles or microparticles, multiple core parts are formed. The cores can be generated during the preparation process or by using polymers or inorganic salts, etc. Form; Figure 5, Figure 9, Figure 13 and Figure 17, when the water-soluble components or non-water-soluble components in the cells or tissue components carried by nanoparticles or micro-particles are distributed inside the nanoparticles or micro-particles, they are located in the formed The outer layer of the inner core; a: the water-soluble components in the inner and surface of nanoparticles or microparticles are all contained in cells or tissue components; Water-insoluble components in tissue components; c: Nanoparticles or microparticles are loaded with water-insoluble components in cells or tissue components, and the surface is loaded with water-soluble components in cells or tissue components; d: Nanoparticles or microparticles contain water-soluble components in cells or tissue components, while surface-loaded components are all water-insoluble components in cells or tissue components; e: Nanoparticles or microparticles contain both The water-soluble components and non-water-soluble components in the loaded cells or tissue components, and the surface of nanoparticles or microparticles also loads the water-soluble components and non-water-soluble components in cells or tissue components; f: nanoparticles or microns The water-soluble components and non-water-soluble components in the cells or tissue components contained inside the particles at the same time, while the surface of the nanoparticles or microparticles only loads the water-soluble components in the cells or tissue components; g: inside the nanoparticles or microparticles The water-soluble components and water-insoluble components in the cell or tissue components are simultaneously contained, while the surface of nanoparticles or microparticles only loads the water-insoluble components in cells or tissue components; h: nanoparticles or The interior of microparticles only contains water-insoluble components in cells or tissue components, while the surface of nanoparticles or microparticles simultaneously loads water-soluble components and insoluble components in cells or tissue components; i: nanoparticles or microparticles The interior of the particles only contains water-soluble components in cells or tissue components, while the surface of nanoparticles or microparticles simultaneously loads water-soluble components and water-insoluble components in cells or tissue components.
图18-图28所示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粒子或微米粒子的结构示意图，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。图18-图19中纳米粒子或微米粒子表面和内部均含有免疫佐剂；图20-图21中免疫佐剂只分布于纳米粒子或微米粒子的内部；图22-图23中纳米粒子或微米粒子只在外表面含有免疫佐剂；图24-图25纳米粒子或微米粒子内部和外表面均无免疫佐剂；图26细胞组分和/或免疫佐剂只分布于纳米粒子或微米粒子内部；图27细胞组分和/或免疫佐剂只分布于纳米粒子或微米粒子外部 ；图28细胞组分和免疫佐剂分别分布于纳米粒子或微米粒子内部或外部。在图18-25中，图18中2.a-2.i，图20中6.a-6.i，图22中10.a-10.i和图24中14.a-14.i纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；图19中3.a-3.i，图20中7.a-7.i，图22中11.a-11.i和图24中15.a-15.i中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的一个内核部分；图19中4.a-4.i，图21中8.a-8.i，图23中12.a-12.i和图25中16.a-16.i纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的多个内核部分；图19中5.a-5.i，图21中9.a-9.i，图23中13.a-13.i和图25中17.a-17.i纳米粒子或微米粒子所包载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部所形成内核的外层； a：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的水溶性成分；b：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的非水溶性成分；c：纳米粒子或微米粒子内部包载的为细胞或组织组分中的非水溶性成分而表面负载的均为细胞或组织组分中的水溶性成分；d：纳米粒子或微米粒子内部包载的为细胞或组织组分中的水溶性成分而表面负载的均为细胞或组织组分中的非水溶性成分；e：纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面也同时负载细胞或组织组分中的水溶性成分和非水溶性成分；f: 纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的水溶性成分；g: 纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的非水溶性成分；h：纳米粒子或微米粒子内部只包载的细胞或组织组分中的非水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；i: 纳米粒子或微米粒子内部只包载的细胞或组织组分中的水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；在图26-28中，a, b和c中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；d, e和f中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的一个内核部分；g，h和i中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的多个内核部分； j，k和l中纳米粒子或微米粒子所包载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部所形成内核的外层； a,
d, g 和j中纳米粒子或微米粒子负载的均为细胞或组织组分中的水溶性成分；b，e，h和k中纳米粒子或微米粒子负载的均为细胞或组织组分中的非水溶性成分；c，f，i和l中纳米粒子或微米粒子同时负载细胞或组织组分中的水溶性成分和非水溶性成分。Fig. 18-Fig. 28 are schematic diagrams showing the structures of nanoparticles or microparticles loaded with water-soluble and water-insoluble cell components actively targeting the target head modification, wherein 1: water-soluble components in cell or tissue components; 2: Water-insoluble components in cells or tissue components; 3: Immunological adjuvants; 4: Nanoparticles or microparticles; 5: The core part of nanoparticles; 6: Targets that can target specific cells or tissues. In Figure 18-Figure 19, the surface and interior of nanoparticles or microparticles contain immune adjuvants; in Figure 20-Figure 21, immune adjuvants are only distributed in the interior of nanoparticles or microparticles; Particles only contain immune adjuvant on the outer surface; Figure 24-Figure 25 has no immune adjuvant inside and outside the nanoparticle or microparticle; Figure 26 cell components and/or immune adjuvant are only distributed inside the nanoparticle or microparticle; Figure 27 The cell components and/or immune adjuvants are only distributed outside the nanoparticles or microparticles; Figure 28 The cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively. In Figures 18-25, 2.a-2.i in Figure 18, 6.a-6.i in Figure 20, 10.a-10.i in Figure 22 and 14.a-14.i in Figure 24 When the water-soluble or non-water-soluble 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. 19, Fig. 7.a-7.i in 20, 11.a-11.i in Figure 22 and 15.a-15.i in Figure 24 are water-soluble components in the cell 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 19, 8.a-8.i in Figure 21, 12.a-12.i in Figure 23 And the water-soluble components or non-water-soluble components in the cells or tissue components loaded by 16.a-16.i nanoparticles or microparticles in Figure 25 are distributed in multiple core parts inside the nanoparticles or microparticles; Figure 19 In 5.a-5.i, 9.a-9.i in Figure 21, 13.a-13.i in Figure 23 and 17.a-17.i in Figure 25 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 non-water-soluble components in the components; in Figures 26-28 In a, b and c, the water-soluble or insoluble components in the cells or tissue components loaded by nanoparticles or microparticles did not form an obvious inner core when they were distributed inside the nanoparticles or microparticles; d, e and The water-soluble components or water-insoluble components in the cells or tissue components loaded by the nanoparticles or microparticles in f are distributed in a core part inside the nanoparticles or microparticles; the nanoparticles or microparticles in g, h and i The water-soluble components or non-water-soluble components in the loaded cells or tissue components are distributed in multiple inner core parts inside the nanoparticles or microparticles; the cells or tissue groups contained in the nanoparticles or microparticles in j, k and l The water-soluble components or non-water-soluble components in the fraction are distributed in the outer layer of the inner core formed inside the nanoparticle or micron particle; a,
The nanoparticles or microparticles in d, 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-soluble components in cells or tissue components Water-insoluble components; c, f, i, and l, nanoparticles or microparticles simultaneously load water-soluble components and water-insoluble components in cell or tissue components.
图29-37分别为实施例1-9中用多种癌症细胞或肿瘤组织制备的纳米疫苗或微米疫苗用于预防或治疗癌症时小鼠肿瘤生长速度和生存期实验结果；a, 纳米疫苗或微米疫苗预防或治疗癌症时的肿瘤生长速度实验结果 （n≥8）； b, 纳米疫苗或微米疫苗预防或治疗其他癌症时的小鼠生存期实验结果（n≥8），每个数据点为平均值±标准误差（mean±SEM）；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；**表示疫苗组与PBS空白对照组相比p＜0.005，有显著性差异；##代表疫苗组与空白纳米粒+细胞裂解物对照组相比p＜0.005，有显著性差异； ***表示疫苗组与PBS空白对照组相比p＜0.0005，有显著性差异；###代表疫苗组与空白纳米粒+细胞裂解物对照组相比p＜0.0005，有显著性差异。 ￥￥￥表示疫苗组与多肽纳米粒组相比p＜0.0005，有显著性差异；ɸ代表疫苗组与对照疫苗组（只含B16F10组分或只含肝癌组织成分）相比p＜0.05，有显著性差异；θ代表疫苗组与无佐剂疫苗组（相比p＜0.05，有显著性差异。Figures 29-37 are respectively the nano-vaccine or micro-vaccine prepared by various cancer cells or tumor tissues in Examples 1-9 for the mouse tumor growth rate and survival experiment results when preventing or treating cancer; a, nano-vaccine or The experimental results of tumor growth rate when micron vaccines prevent or treat cancer (n≥8); b, the experimental results of mouse survival time when nano vaccines or micron vaccines prevent or treat other cancers (n≥8), each data point is 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; ** indicates that the vaccine group and Compared with the PBS blank control group, p<0.005, there is a significant difference; ## represents the vaccine group and the blank nanoparticle + cell lysate control group, p<0.005, there is a significant difference; *** represents the vaccine group and the PBS blank control group Compared with the control group, p<0.0005, there is a significant difference; ### represents that the vaccine group has a significant difference, p<0.0005, compared with the blank nanoparticle + cell lysate control group. ￥￥￥means p<0.0005, there is a significant difference between the vaccine group and the polypeptide nanoparticle group; ɸ represents p<0.05, there is Significant difference; θ represents a significant difference between the vaccine group and the non-adjuvanted vaccine group (p<0.05 compared.
本发明公开了一种纳米级或微米级的负载一种以上癌症细胞和/或肿瘤组织全细胞组分的广谱癌症疫苗系统及其应用预防或治疗癌症。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 broad-spectrum cancer vaccine system loaded with more than one cancer cell and/or whole cell components of tumor tissue at the nanometer or micrometer level and its application to prevent or treat cancer. 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.
本发明在将癌细胞和/或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 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 in the aqueous solution containing a solubilizing agent. In the solubilization solution, all the cellular components can be converted into components that can be dissolved in aqueous solution and then loaded inside and outside the nanoparticles or microparticles to prepare nano-vaccine or micro-vaccine for cancer prevention and treatment . 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，从而提高了纳米粒子或微米粒子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 Comprehensiveness and immunogenicity of antigenic substances or components loaded on nanoparticles or microparticles.
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.
细胞组分中水溶性部分和非水溶性部分囊括了整个细胞的成分和组分。其中与正常细胞成分相同未突变的蛋白质、多肽和基因因为自身免疫系统发育过程中所产生的免疫耐受不会引起免疫反应；而因为癌症产生的基因、蛋白质和多肽的突变因为没有自身免疫系统发育过程中所产生的免疫耐受因而具有免疫原性且可激活机体针对癌细胞的免疫反应。利用全细胞组分中这些因为疾病突变而产生的具有癌细胞特异性免疫原性的物质即可用于癌症的预防和治疗。The water-soluble part and the water-insoluble part of the cell components include the components and components of the whole cell. 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 generated during the development of the autoimmune system; while the mutations of genes, proteins and polypeptides produced by cancer will not cause immune responses due to the absence of an autoimmune system The immune tolerance developed during development is thus immunogenic and activates the body's immune response against cancer cells. The substances with specific immunogenicity of cancer cells produced by disease mutations in the whole cell components can be used for the prevention and treatment of cancer.
本发明所述纳米疫苗和/或微米疫苗系统可用于制备预防和/或治疗癌症的疫苗，其制备过程及应用领域如图1所示。在制备时可裂解细胞或组织后先分别收集水溶性组分和水不溶性组分并分别制备纳米疫苗或微米疫苗；或者也可以直接采用含有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 nano-vaccine and/or micro-vaccine system of the present invention can be used to prepare a vaccine for preventing and/or treating cancer, and its preparation process and application fields are shown in FIG. 1 . 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.
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、酶处理或（和）变性处理直接制备纳米疫苗或微米疫苗。本发明部分实施例中，肿瘤组织细胞在裂解前经过了灭活或（和）变性处理，在实际使用过程中也可以在细胞裂解后做灭活、酶处理、或（和）变性处理，或者也可以细胞裂解前和裂解后均做灭活、酶处理或（和）变性处理；本发明部分实施例中细胞裂解前或（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, enzyme treatment 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, and may be inactivated, enzymatically treated, or (and) denatured after cell lysing, or Inactivation, enzyme treatment or (and) denaturation treatment can also be performed before and after cell lysis; in some embodiments of the present invention, the inactivation or (and) denaturation treatment method before or (and) after cell lysis 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 actual use process. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific conditions.
本发明所述广谱癌症疫苗系统，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 surface of the broad-spectrum cancer vaccine system of the present invention may not be connected with a target head with active targeting function or be connected with a target head with active targeting function.
本发明所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的疫苗系统的结构示意图如图2-图28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和/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和/或微米粒子。The structural diagrams of the vaccine system for preventing or treating cancer based on various cancer cells and/or whole cell components of tumor tissues according to the present invention are shown in FIGS. 2-28 . In actual use, only nanoparticles and/or microparticles of a specific structure may be used, or two or more nanoparticles and/or microparticles of different structures may be used simultaneously.
在实施例中，免疫增强剂包载于纳米粒子或微米粒子内并同时负载于纳米粒子或微米粒子表面，在实际使用过程中免疫增强剂也可只包载于纳米粒子或微米粒子内，或者只负载于纳米粒子或微米粒子表面，或者不加入免疫增强剂。In an embodiment, the immunopotentiator is entrapped in nanoparticles or microparticles and simultaneously loaded on the surface of nanoparticles or microparticles. In actual use, the immunopotentiator can also be entrapped only in nanoparticles or microparticles, or It is only loaded on the surface of nanoparticles or microparticles, or no immune enhancer is added.
在一些实施例中，本发明先将细胞组分中的可溶于纯水的水溶性部分或（和）非水溶性部分经增溶剂进行增溶后包载于纳米粒子或微米粒子内，同时负载免疫增强剂；然后，将细胞组分中的水溶性部分或（和）非水溶性部分负载于纳米粒子表面，同时负载有免疫增强剂。这样就使得纳米粒子或微米粒子中细胞的水溶性组分或非水溶性组分的负载能力可以达到最大。在实际应用中，也可以直接采用含有增溶剂的增溶液（如8M尿素水溶液或6M盐酸胍水溶液）直接裂解细胞或组织并直接溶解全细胞组分，尔后以此制备纳米疫苗或微米疫苗。In some embodiments, in the present invention, the water-soluble part or (and) water-insoluble part soluble in pure water in the cell component is firstly solubilized by a solubilizer, and then encapsulated in nanoparticles or microparticles, and at the same time Immunopotentiator is loaded; then, the water-soluble part or (and) non-water-soluble part of the cell component is loaded on the surface of the nanoparticle, and the immunopotentiator is loaded at the same time. This maximizes the loading capacity of the water-soluble or water-insoluble components of the cells in the nanoparticles or microparticles. In practical applications, it is also possible to directly use a solubilizing solution containing a solubilizing agent (such as 8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution) to directly lyse cells or tissues and directly dissolve whole cell components, and then prepare nano-vaccine or micro-vaccine.
在本发明所述制备纳米疫苗及微米疫苗的方法为常用制备方法。在一些实施方案中，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为有机高分子聚乳酸-羟基乙酸共聚物（PLGA）分子量为24KDa-38KDa, 所采用的免疫佐剂为 poly(I:C)、卡介苗（BCG）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 nano-vaccine and micro-vaccine described in the present invention is a common preparation method. In some embodiments, the double emulsion method in the solvent volatilization method is used to prepare the nano vaccine. Adjuvants are poly(I:C), Bacillus Calmette-Guerin (BCG) 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 preparation materials used, types and concentrations of immune adjuvants, 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裂解物中的各组分以及免疫增强佐剂poly(I:C)、BCG或CpG；癌细胞裂解物中的各组分在制备时分别为水溶性组分或者是溶于8M尿素中的原非水溶性组分。水相溶液所含有来自癌细胞的水溶性组分的浓度或者是来自癌细胞的溶于增溶剂的原非水溶性组分的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于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 may contain each component in the cancer cell lysate and the immune enhancing adjuvant poly(I:C), BCG or CpG; each component in the cancer cell lysate is prepared as The water soluble component or the original water insoluble component dissolved in 8M urea. The concentration of the water-soluble components from cancer cells contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in the solubilizing agent from cancer cells, that is, the first predetermined concentration requires that the protein polypeptide concentration is 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.
在一些实施例中，水相溶液含有肿瘤组织裂解物中的各组分以及免疫增强佐剂poly(I:C)，BCG或CpG；肿瘤组织裂解物中的各组分在制备时分别为水溶性组分或者是溶于8M尿素中的原非水溶性组分或者全细胞组分均溶解于8M尿素或6M盐酸胍等增溶剂中。水相溶液所含有得来自肿瘤组织的水溶性组分的浓度或者是来自肿瘤组织的溶于8M尿素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 tumor tissue lysate and the immune enhancing adjuvant poly(I:C), BCG or CpG; each component in the tumor tissue lysate is water-soluble respectively during preparation The active components or the original water-insoluble components dissolved in 8M urea or the whole cell components are all dissolved in solubilizers such as 8M urea or 6M guanidine hydrochloride. The concentration of the water-soluble component from the tumor tissue contained in the aqueous phase solution or the concentration of the original water-insoluble component dissolved in 8M urea from the tumor tissue, that is, the first predetermined concentration requires that the protein polypeptide concentration is 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或修饰的额PLGA，是由于该材料为生物可降解材料且已被FDA批准用作药物敷料。研究表明PLGA具有一定的免疫调节功能，因而适合作为疫苗制备时的辅料。In the present invention, PLGA or modified frontal PLGA was chosen because the material is biodegradable and has been approved by the FDA for use as a drug dressing. 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.
优选的，水相溶液为裂解物组分溶液时，其中蛋白质和多肽的浓度大于1 ng/mL，优选1 mg/mL～100 mg/mL；水相溶液为裂解物组分/免疫佐剂溶液时，其中蛋白质和多肽的浓度大于1
ng/mL，优选1 mg/mL～100
mg/mL，免疫佐剂的浓度大于0.01 ng/mL，优选0.01
mg/mL～20 mg/mL。高分子材料有机相溶液中，溶剂为DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等，优选二氯甲烷；高分子材料的浓度为0.5
mg/mL～5000 mg/mL，优选为100
mg/mL。第一乳化剂溶液优选为聚乙烯醇水溶液，浓度为10 mg/mL～50 mg/mL，优选20 mg/mL。第二乳化剂溶液优选为聚乙烯醇水溶液，浓度为1 mg/mL～20
mg/mL，优选5 mg/mL。分散液为PBS缓冲液或生理盐水或纯水。Preferably, when the aqueous phase solution is a lysate component solution, the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL～100 mg/mL; the aqueous phase solution is a lysate component/immune adjuvant solution When the concentration of protein and peptide is greater than 1
ng/mL, preferably 1 mg/mL～100
mg/mL, the concentration of immune adjuvant is greater than 0.01 ng/mL, preferably 0.01
mg/mL～20 mg/mL. In the polymer material organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the polymer material is 0.5
mg/mL～5000 mg/mL, preferably 100
mg/mL. The first emulsifier solution is preferably an aqueous solution of polyvinyl alcohol, with a concentration of 10 mg/mL-50 mg/mL, preferably 20 mg/mL. The second emulsifier solution is preferably an aqueous polyvinyl alcohol solution with a concentration of 1 mg/mL～20
mg/mL, preferably 5 mg/mL. The dispersion liquid is PBS buffer solution or physiological saline or pure water.
步骤2，将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。优选的，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50 rpm，搅拌时间大于1分钟，比如搅拌速度为50 rpm～1500 rpm，搅拌时间为0.1小时～24小时；超声处理时，超声功率大于5W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于5 psi，比如20psi～100psi，使用高剪切均质机时转速大于100 rpm，比如1000
rpm～5000 rpm；使用微流控处理流速大于0.01
mL/min, 比如0.1 mL/min-100 mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米和/或微米粒子大小，过大或过小都会带来粒径大小的变化。Step 2, subjecting the mixed liquid obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5W, the time is greater than 0.1 seconds, such as 2 to 200 seconds; use a high-pressure/ultra-high pressure homogenizer or a high-shear homogenizer for homogenization, and the pressure is greater than 5 psi when using a high-pressure/ultra-high pressure homogenizer, such as 20psi～ 100psi, when using a high shear homogenizer, the speed is greater than 100 rpm, such as 1000
rpm～5000 rpm; use microfluidics to process flow rate greater than 0.01
mL/min, such as 0.1 mL/min-100 mL/min. Ultrasound or stirring or homogenization treatment or microfluidic treatment for nanometerization and/or micronization, the length of ultrasonic time or stirring speed or homogenization treatment pressure and time can control the size of the prepared nanometer and/or micron particles, too large or too large Small will bring about changes in particle size.
步骤3，将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。该步骤是为了进行纳米化或微米化，超声时间长短或搅拌速度及时间能控制制备的纳米粒子或微米粒子大小，过长或过短都会带来粒径大小的变化，为此，需要选择合适的超声时间。在本发明中，超声时间大于0.1秒，比如2～200秒，搅拌速度大于50rpm，比如50 rpm～500 rpm，搅拌时间大于1分钟，比如60～6000秒。优选的，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50 rpm，搅拌时间大于1分钟，比如搅拌速度为50 rpm～1500
rpm，搅拌时间为0.5小时～5小时；超声处理时，超声功率为50W～500W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于20psi，比如20psi～100psi，使用高剪切均质机时转速大于1000 rpm，比如1000
rpm～5000 rpm；使用微流控处理流速大于0.01
mL/min, 比如0.1 mL/min-100 mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米或微米粒子大小，过大或过小都会带来粒径大小的变化。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 homogeneous treatment or microfluidic control deal with. In this step, the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be nanometerized or micronized by ultrasonication or stirring. 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 or microparticles. Too long or too short will bring about changes in particle size. For this reason, it is necessary to choose a suitable the ultrasound 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 60-6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm.
rpm, the stirring time is 0.5 hours to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W, and the time is greater than 0.1 seconds, such as 2 to 200 seconds; Homogenizer, when using a high-pressure/ultrahigh-pressure homogenizer, the pressure is greater than 20psi, such as 20psi~100psi, and when using a high-shear homogenizer, the speed is greater than 1000 rpm, such as 1000
rpm～5000 rpm; use microfluidics to process flow rate greater than 0.01
mL/min, such as 0.1 mL/min-100 mL/min. Ultrasound or stirring or homogenization treatment or microfluidic treatment for nanometerization or micronization, the length of ultrasonic time or stirring speed or homogenization treatment pressure and time can control the size of the prepared nanometer or micron particles, too large or too small will bring Changes in particle size.
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粒子或微米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。同样地，本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒或微米粒。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. the
In order to control the size of nanoparticles or microparticles during specific implementation, the ratio of the second predetermined volume to the third predetermined volume may be adjusted. 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，并进行搅拌直至满足预定搅拌条件。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 stirring until predetermined stirring conditions are met.
本步骤中，乳化剂水溶液依然为PVA。In this step, the emulsifier aqueous solution is still 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。The fourth predetermined concentration is 5 mg/mL, and 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，本步骤的预定搅拌条件为直至有机溶剂挥发完成，也即步骤1中的二氯甲烷挥发完成。In the present invention, the predetermined stirring condition of this step is until the organic solvent is volatilized, that is, the dichloromethane in step 1 is volatilized.
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于100rpm的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS（或生理盐水）中。Step 5, after centrifuging the mixed solution that meets the predetermined stirring conditions in step 4 at a speed greater than 100 rpm for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate 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 the subsequent adsorption of cancer cell lysate on the surface of nanoparticles or microparticles can be performed directly. Related experiments.
在本发明一些实施方案中，步骤5所得沉淀重新混悬于含有冻干保护剂的水溶液中时需进行冷冻干燥，再冷冻干燥以后再进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验。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 correlation of the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles is carried out. experiment.
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 concentration of the lyoprotectant in this step is 4% by mass, which is set so as not to 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）中的含纳米粒的混悬液或者采用第六预定体积的PBS（或生理盐水）重悬步骤6得到的冷冻干燥后的含有纳米粒或微米粒和冻干保护剂的冻干物质，与第七预定体积的水溶性组分或者溶于8M尿素中的原非水溶性组分混合后即得纳米疫苗或微米疫苗。Step 7, resuspending the nanoparticle-containing suspension obtained in step 5 with the sixth predetermined volume in PBS (or normal saline) or using the sixth predetermined volume of PBS (or normal saline) to resuspend the suspension obtained in step 6 The freeze-dried freeze-dried substance containing nanoparticles or microparticles and a freeze-drying protective agent is mixed with the seventh predetermined volume of water-soluble components or the original water-insoluble components dissolved in 8M urea to obtain nano-vaccine or micron vaccines.
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混悬液体积为10 mL时，含有癌细胞裂解物或含有肿瘤组织裂解物中的水溶性组分或者溶于8M尿素中的原非水溶性组分的体积与为1 mL。在实际使用时可将二者体积和比例根据需要进行调整。In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, it contains cancer cell lysates or contains water-soluble components in tumor tissue lysates or original water-insoluble components dissolved in 8M urea. The volume of the components is 1 mL. The volume and ratio of the two can be adjusted as required during actual use.
在本发明中，所采用的含有癌细胞裂解物或含有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）或CpG，且poly(I:C)、BCG或CpG的浓度为大于1 ng/mL。In the present invention, the used water-soluble components containing cancer cell lysates or tumor tissue lysates or the original non-water-soluble components dissolved in 8M urea contain poly(I:C), Bacillus Calmette-Guerin (BCG) 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，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 the nano-vaccine or micro-vaccine is nanometer or micrometer, which can ensure that the vaccine is phagocytized by antigen-presenting cells, and 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裂解物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裂解物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 cancer cell lysates or tumor tissue lysates, and any other components that can make cancer cell lysates or tumor tissue lysates 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 solubilizer can be used to dissolve both water-soluble 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裂解物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裂解物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 cell lysates or tumor tissue lysates, and any other components that can make cancer cells The original water-insoluble components in the cell lysate or tumor tissue lysate are dissolved in the urea concentration or the guanidine hydrochloride concentration of the aqueous solution; or the water-soluble components and the water-insoluble components are simultaneously dissolved using 8M urea aqueous solution.
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，癌细胞裂解物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粒子内部和吸附在纳米粒子表面，在实际使用时，癌细胞裂解物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负载到纳米粒子内部或负载到纳米粒子表面；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。负载方式包括化学键结合和/或非化学键吸附。In addition, in the embodiment of the present invention, the water-soluble components in the cancer cell lysate or the tumor tissue lysate or the original water-insoluble components dissolved in 8M urea are respectively loaded inside the nanoparticles and adsorbed on the surface of the nanoparticles, In actual use, the water-soluble components in the cancer cell lysate or tumor tissue lysate and the original water-insoluble components dissolved in 8M urea can also be mixed and then loaded into the interior of the nanoparticles or loaded onto the surface of the nanoparticles; or It is also possible to use 8M urea to simultaneously dissolve the water-soluble component and the water-insoluble component and then entrap the inside of the nanoparticle or microparticle and/or adsorb on the surface of the nanoparticle or microparticle. Loading methods include chemical bonding and/or non-chemical bonding adsorption.
另外，在本发明中，采用poly(I:C)、锰佐剂、卡介苗（BCG）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、铝佐剂、羊毛脂、植物油、内毒素、脂质体佐剂、GM-CSF、MF59、双链RNA、双链DNA、CAF01、人参、黄芪等中药有效成分。In addition, in the present invention, poly(I:C), manganese adjuvant, Bacillus Calmette-Guerin (BCG) and CpG are used as immune adjuvants. In practice, no immune adjuvant or any other immune adjuvant with immune enhancing function can be added. Agents, such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl dipeptide, Mycobacterium phlei, polyclonal A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virus Body, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, neonatal bovine liver active peptide, imiquimod, polysaccharide, curcumin, immune adjuvant poly ICLC, Corynebacterium pumilus vaccine, hemolytic streptococcus preparation , coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum adjuvant, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM - CSF, MF59, double-stranded RNA, double-stranded DNA, 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和/或微米疫苗。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 and/or micro-vaccine according to the actual situation.
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。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尺寸粒子或微米尺寸粒子结构、制备方法、疾病治疗时的使用策略等仅为代表性方法，其他纳米尺寸粒子或微米尺寸粒子结构、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 nano-sized particle or micron-sized particle structure, preparation method, and use strategy during disease treatment mentioned in the embodiments of the present invention are only representative methods, and other nano-sized particles or micron-sized particle structures, preparation methods, disease prevention or The use strategy during treatment 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 黑色素瘤肿瘤组织和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：本实施例以小鼠黑色素瘤为癌症模型来说明如何制备负载有黑色素瘤肿瘤组织和肺癌肿瘤组织的全细胞组分的纳米疫苗，并应用该疫苗预防黑色素瘤。Example 1 The whole cell components of melanoma tumor tissue and lung cancer tumor tissue are loaded inside and on the surface of nanoparticles for the prevention of melanoma: this example uses mouse melanoma as a cancer model to illustrate how to prepare melanoma tumor tissue and whole-cell components of lung cancer tumor tissue as a nanovaccine, and apply the vaccine to prevent melanoma.
本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。首先裂解B16F10黑色素瘤肿瘤组织和LLC肺癌肿瘤组织以制备肿瘤组织的水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以Polyinosinic-polycytidylic acid (poly(I:C))为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织的水溶性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。In this example, B16F10 mouse melanoma cells were used as the cancer model. Firstly, the B16F10 melanoma tumor tissue and the LLC lung cancer tumor tissue were lysed to prepare the water-soluble fraction and the water-insoluble fraction of the 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 the water-soluble component and the water-insoluble component loaded with tumor tissue by the solvent evaporation method. Divided nano-vaccine. The nanovaccine was then employed to prevent melanoma.
（1）肿瘤组织的裂解及各组分的收集：在每只C57BL/6小鼠背部皮下接种1.5×10
5个B16-F10 细胞或者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适量纯水并反复冻融5次，并可伴有超声以破坏裂解组织细胞。待组织细胞裂解后，将裂解物以5000g的转速离心3分钟并取上清液即为可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。将来自B16-F10肿瘤组织的和来自LLC肺癌肿瘤组织的水溶性组分和溶解于8M尿素中的原非水溶性组分分别按照1:1的比例混合即为制备疫苗的原料来源。在对照纳米疫苗中，本实施例采用负载等比例水溶性多肽B16-M20
(Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24
(Dag1, TAVITPPTTTTKKARVSTPKPATPSTD)，B16-M27
(REGVELCPGNKYEMRRHGTTHSLVIHD) 和 8M尿素增溶的水不溶性多肽 B16-M05(Eef2, FVVKAYLPVNESFAFTADLRSNTGGQA), B16-M46
(Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ), and TRP2:180-188(SVYDFFVWL)。
(1) Lysis of tumor tissue and collection of various components: subcutaneously inoculate 1.5×10 5 B16-F10 cells or 2×10 6 LLC lung cancer cells on the back of each C57BL/6 mouse, and when the tumor grows to the volume, respectively The mice were sacrificed when the size was about 1000 mm 3 and the tumor tissues were removed. After the tumor tissue was cut into pieces, it was ground, and an appropriate amount of pure water was added through a cell strainer, followed by repeated freezing and thawing 5 times, accompanied by ultrasound to destroy and lyse the tissue cells. After the tissue cells are lysed, centrifuge the lysate at a speed of 5000g for 3 minutes and take the supernatant, which is the water-soluble component soluble in pure water; The water-insoluble components of pure water were converted to be soluble in 8M aqueous urea solution. The water-soluble components from B16-F10 tumor tissue and LLC lung cancer tumor tissue and the original water-insoluble components dissolved in 8M urea were mixed in a ratio of 1:1, which was the source of raw materials for preparing vaccines. In the control nano-vaccine, this embodiment adopts water-soluble polypeptide B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM) in equal proportions, B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M27 (REGVELCPGNKYEMRRHGTTHSLVIHD) and 8M urea solubilized water-insoluble polypeptide B16 -M05 (Eef2, FVVKAYLPVNESFAFTADLRSNTGGQA), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ), and TRP2:180-188 (SVYDFFVWL).
（2）纳米疫苗的制备：本实施例中纳米疫苗及作为对照的空白纳米粒以及负载多种多肽的纳米粒采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫佐剂为poly(I:C)
且poly(I:C)既分布于纳米粒子内部也负载于纳米粒子表面。制备方法如前所述。负载全细胞组分的纳米疫苗平均粒径为320nm左右，纳米疫苗表面电位为-5mV左右；每1mg PLGA纳米粒子约负载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270nm左右，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)。负载多肽的纳米粒制备方法同制备负载全细胞组分的制备方法，所使用的poly(I:C)的量也相同，多肽纳米粒粒径约为310nm, 每1mg
PLGA纳米粒子约负载150μg多肽组分。以来自B16-F10肿瘤组织的水溶性组分和溶解于8M尿素中的原非水溶性组分为原料来源制备只负载B16F10肿瘤组织全细胞组分的疫苗。(2) Preparation of nano-vaccine: In this example, the nano-vaccine, the blank nano-particles used as the control, and the nanoparticles loaded with various polypeptides were prepared by the double emulsion method in the solvent evaporation method, and the molecular weight of the nano-particle preparation material PLGA used was 24KDa-38KDa, the immune adjuvant used is poly(I:C)
And poly(I:C) is not only distributed inside the nanoparticles but also loaded on the surface of the nanoparticles. The preparation method is as described above. The average particle size of the nano-vaccine loaded with whole cell components is about 320nm, and the surface potential of the nano-vaccine is about-5mV; every 1mg PLGA nano-particle is about loaded with 180 μg protein or polypeptide component, and the poly(I: C) The immune adjuvant is about 0.01 mg in total and half inside and outside. The particle size of blank nanoparticles is about 270nm. 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 same amount of poly(I:C) as nanovaccine was adsorbed on the surface. The preparation method of polypeptide-loaded nanoparticles is the same as the preparation method of preparing whole-cell components, and the amount of poly(I:C) used is also the same. The particle size of polypeptide nanoparticles is about 310nm, and every 1mg
PLGA nanoparticles are loaded with about 150 μg of polypeptide components. The water-soluble component from B16-F10 tumor tissue and the original water-insoluble component dissolved in 8M urea were used as raw materials to prepare the vaccine loaded only with the whole cell component of B16F10 tumor tissue.
（3）纳米疫苗用于癌症的预防：本研究对照组分别是PBS组和空白纳米粒+游离组织裂解物组。选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。(3) Nano-vaccine for the prevention of cancer: the control groups in this study were the PBS group and the blank nanoparticle+free tissue lysate group. Select 6-8-week-old female C57BL/6 as model mice to prepare melanoma-bearing mice.
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内部和表面都负载水溶性成分的2mg PLGA纳米疫苗和200μL内部和表面都负载原非水溶性成分的2mg PLGA纳米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10
5个B16F10细胞。
The administration regimen of the nanovaccine group was as follows: 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components on the inside and on the surface were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation and 200 μL of 2 mg PLGA nano-vaccine loaded with original water-insoluble components on the inside and on the surface; on day 0, 1.5×10 5 B16F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse.
PBS对照组方案如下：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射400μL PBS；在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。空白纳米粒+游离裂解物对照组：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射400μL 空白纳米粒和与疫苗负载的等量的游离裂解物；空白纳米粒和游离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。
The protocol of the PBS control group is as follows: 400 μL PBS was injected subcutaneously on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation; on the 0th day, 1.5×105 mice were subcutaneously inoculated on the lower right back of each mouse B16F10 cells. Blank nanoparticles + free lysate control group: 400 μL of blank nanoparticles and the same amount of free lysate loaded with the vaccine were subcutaneously injected on days 49, 42, 35, 28 and 14 before inoculation of melanoma; blank 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.
多肽纳米粒组给药方案如下：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内部和表面都负载水溶性成分的2mg PLGA纳米疫苗和200μL内部和表面都负载原非水溶性成分的2mg PLGA纳米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10
5个B16F10细胞。
The dosage regimen of the polypeptide nanoparticle group was as follows: 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components on the inside and on the surface were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. and 200 μL of 2 mg PLGA nano-vaccine loaded with the original water-insoluble components both inside and on the surface; on day 0, 1.5×10 5 B16F10 cells were subcutaneously inoculated on the lower right side of the 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29所示，负载B16F10和LLC混合肿瘤组织全细胞组分的疫苗处理组小鼠的肿瘤在接种后全部消失；只负载B16F10肿瘤组织的疫苗处理组约70%小鼠的肿瘤在接种后消失；负载多肽的纳米粒子组只有25%左右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产速度很快。综上所述，本发明所述的负载多种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良好的预防效果。(4) Experimental results: As shown in Figure 29, the tumors of mice in the vaccine treatment group loaded with B16F10 and LLC mixed tumor tissue whole cell components all disappeared after inoculation; about 70% of the tumors in the vaccine treatment group loaded with B16F10 tumor tissue were smaller The tumors of the mice disappeared after inoculation; only about 25% of the tumors of the mice in the peptide-loaded nanoparticle group disappeared after inoculation. In contrast, the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger and produced rapidly. In summary, the nano-vaccine loaded with water-soluble components and water-insoluble components of various tumor tissues according to the present invention has a good preventive effect on melanoma.
实施例2 黑色素瘤和肺癌细胞水溶性细胞组分负载于微米粒子内部和表面用于黑色素瘤的预防：本实施例以小鼠黑色素瘤为癌症模型来说明如何制备只负载有黑色素瘤和肺癌细胞组分中水溶性部分的微米疫苗，并应用该疫苗预防黑色素瘤。Example 2 Water-soluble cell components of melanoma and lung cancer cells are loaded on the inside and surface of microparticles for the prevention of melanoma: This example uses mouse melanoma as a cancer model to illustrate how to prepare only melanoma and lung cancer cells A micron vaccine of the water-soluble part in the composition, and apply the vaccine to prevent melanoma.
本实施例中，首先裂解B16F10黑色素瘤和LLC肺癌细胞以制备水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA（38KDa-54KDa）为微米粒子骨架材料，以poly(I:C)为免疫佐剂采用溶剂挥发法制备负载有全细胞的水溶性组分的微米疫苗。然后采用该微米疫苗来预防黑色素瘤。In this example, B16F10 melanoma and LLC lung cancer cells were firstly lysed to prepare water-soluble fractions and water-insoluble fractions. Then, the organic polymer material PLGA (38KDa-54KDa) was used as the micron particle skeleton material, and poly(I:C) was used as the immune adjuvant to prepare the micron vaccine loaded with the whole cell water-soluble component by the solvent evaporation method. The micron vaccine was then used to prevent melanoma.
（1）癌细胞的裂解及各组分的收集：收集一定量的B16F10细胞或LLC细胞，去除培养基后采用-20℃冷冻，加一定量超纯水后反复冻融3次以上，并可伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以3000g的转速离心5min取上清液即为B16F10黑色素瘤或LLC肺癌细胞中可溶于纯水的水溶性组分。上述所得来源于两种癌细胞裂解物的水溶性组分按1:1混合即为制备微米疫苗的抗原来源。(1) Lysis of cancer cells and collection of various components: collect a certain amount of B16F10 cells or LLC cells, remove the culture medium and freeze at -20°C, add a certain amount of ultrapure water and freeze-thaw more than 3 times repeatedly. Accompanied by sonication to disrupt lysed cells. 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 soluble in pure water in B16F10 melanoma or LLC lung cancer cells. The above-mentioned water-soluble components derived from two kinds of cancer cell lysates are mixed at a ratio of 1:1, which is the antigen source for preparing micron vaccines.
（2）微米疫苗的制备：本实施例中制备微米疫苗及作为对照的空白微米粒采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料为PLGA，所采用的免疫佐剂为 CPG 且CPG既分布于微米粒子内部也负载于微米粒子表面。制备方法如前所述。在微米粒子表面负载细胞组分和免疫佐剂后所得微米疫苗粒径为1.40 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210μg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1.20 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 volatilization method, the micron particle preparation material used is PLGA, and the immune adjuvant used is CPG And the CPG is not only distributed inside the microparticles but also loaded on the surface of the microparticles. The preparation method is as described above. The particle size of the micron vaccine obtained after loading cell components and immune adjuvant on the surface of the micron particle is about 1.40 μm, and the average surface potential of the micron particle is about -5mV. Each 1 mg of PLGA microparticles is loaded with 210 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, with half and half inside. The particle size of the blank microparticles is about 1.20 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。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微米疫苗；在第0天给每只小鼠背部右下方皮下接种1.5×10
5个B16F10细胞。PBS空白对照组方案如下：在接种黑色素瘤之前第28天、第21天、第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。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。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。
(3) Micron vaccine for cancer prevention: select 6-8 week old female C57BL/6 to prepare melanoma tumor-bearing mice. The scheme of the Micron vaccine group was as follows: 400 μL of 4 mg PLGA Micron vaccine loaded with water-soluble components in cancer cell lysates were subcutaneously injected on the 28th day, 21st day, and 14th day before the melanoma inoculation; 1.5× 105 B16F10 cells were inoculated subcutaneously in the lower right lower back of each mouse. The protocol for the PBS blank control group was as follows: 400 μL PBS was subcutaneously injected on the 28th day, 21st day, and 14th day before the inoculation of melanoma; on the 0th day, 1.5× 105 B16F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse. cell. 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. On day 0, 1.5× 105 B16F10 cells were subcutaneously inoculated into the lower right lower 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。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30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。由此可见，本发明所述的负载黑色素瘤和肺癌细胞水溶性组分的微米疫苗对黑色素瘤具有预防效果。(4) Experimental results: As shown in Figure 30, 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. It can be seen that the micro-vaccine loaded with water-soluble components of melanoma and lung cancer cells according to the present invention has a preventive effect on melanoma.
实施例3 肺癌肿瘤组织和肝癌肿瘤组织裂解组分负载于微米粒子内部和表面用于肝癌的预防：本实施例以如何制备负载有肝癌和肺癌肿瘤组织裂解物组分的微米疫苗，并应用该疫苗预防肝癌来说明如何使用肿瘤组织制备的疫苗预防肝癌。Example 3 Lung cancer tumor tissue and liver cancer tumor tissue lysate components are loaded inside and on the surface of microparticles for the prevention of liver cancer: This example is how to prepare micron vaccines loaded with liver cancer and lung cancer tumor tissue lysate components, and apply this Vaccines Prevent Liver Cancer to illustrate how vaccines prepared from tumor tissue can be used to prevent liver cancer.
本实施例中，将小鼠肝癌和肺癌肿瘤组织裂解组分按1:3的比例负载于纳米粒子内部和表面以制备微米疫苗。首先取得小鼠肺癌和肝癌肿瘤组织并将其裂解以制备瘤块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 （24KDa-38KDa）为微米粒骨架材料，以poly(I:C)为免疫佐剂制备微米疫苗。然后采用该微米疫苗来预防Hepa
1-6 肝癌荷瘤小鼠体内的肿瘤。In this example, mouse liver cancer and lung cancer tumor tissue lysates were loaded on the interior and surface of nanoparticles at a ratio of 1:3 to prepare micron vaccines. Firstly, the mouse lung cancer and liver cancer tumor tissues were obtained and lysed to prepare the water-soluble fraction of the tumor mass tissue and the original water-insoluble fraction dissolved in 8M urea. Then, PLGA (24KDa-38KDa) is used as the micron particle skeleton material, and poly(I:C) is used as the immune adjuvant to prepare the micron vaccine. The micron vaccine is then used to prevent Hepa
1-6 Tumors in HCC tumor-bearing mice.
（1）肿瘤组织的裂解及各组分的收集：在每只C57BL/6小鼠腋下皮下接种2×10
6个Hepa 1-6 细胞或者2×10
6个LLC肺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000 mm
3时处死小鼠并摘取肿瘤组织。后续处理方法同实施例1。
(1) Lysis of tumor tissue and collection of various components: Inoculate 2×10 6 Hepa 1-6 cells or 2×10 6 LLC lung cancer cells subcutaneously in the armpit of each C57BL/6 mouse. When the inoculated tumors grew to a volume of about 1000 mm 3 , the mice were sacrificed and the tumor tissues were harvested. Subsequent processing method is the same as embodiment 1.
（2）微米疫苗的制备：方法同实施例2。(2) Preparation of micron vaccine: the method is the same as in Example 2.
（3）微米疫苗用于癌症的预防：选取6-8周的雌性C57BL/6制备 Hepa 1-6 肝癌荷瘤小鼠。(3) Micron vaccine for cancer prevention: 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内部和表面都负载水溶性成分的2mg PLGA微米疫苗和200μL内部和表面都负载原非水溶性成分的2mg PLGA微米疫苗。在第0天给每只小鼠右腋下皮下接种2×10
6个 Hepa 1-6 肝癌细胞。 PBS空白对照组方案如下：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射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。空白微米粒+游离裂解物对照组：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL 空白微米粒和与疫苗所负载的等量的游离裂解物；空白微米粒和游离细胞裂解物注射在不同部位。在第0天给每只小鼠右腋下皮下接种2×10
6个 Hepa 1-6 肝癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
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 micron vaccine loaded with water-soluble components on the inside and on the surface and 200 μL of original non- 2mg PLGA micron vaccine of water soluble composition. 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 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. Blank microparticles+free lysate control group: Subcutaneously inject 400 μL of blank microparticles and the same amount of vaccine-loaded Free lysate; blank microparticles and free cell lysate 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. In the experiment, the mouse tumor growth monitoring method was the same as above.
（4）实验结果：如图31所示，PBS对照组以及空白微米粒+组织裂解物对照组小鼠的肝癌肿瘤生长均较快。微米疫苗给药组小鼠在接种肿瘤后肿瘤消失。由此可见，本发明所述的负载肺癌肿瘤组织和肝癌肿瘤组织裂解物中水溶性组分和非水溶性组分的微米疫苗对肝癌具有预防效果。(4) Experimental results: As shown in Figure 31, the liver cancer tumors in the PBS control group and the blank microparticle + tissue lysate control group grew faster. The tumors disappeared in mice in the Micron vaccine administration group after inoculation. It can be seen that the micro-vaccine loaded with water-soluble components and non-water-soluble components in the lysate of lung cancer tumor tissue and liver cancer tumor tissue according to the present invention has a preventive effect on liver cancer.
实施例4 肺癌和肝癌肿瘤组织全细胞组分负载于纳米粒子内部用于肝癌的预防：本实施例以小鼠肝癌为癌症模型来说明如何制备负载有肺癌和肝癌肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗预防肝癌。首先裂解肺癌和肝癌肿瘤组织以制备全细胞组分的水溶性组分和非水溶性组分并按1:2的比例混合。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负载有肺癌瘤块和肝癌瘤块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肝癌。Example 4 Whole cell components of lung cancer and liver cancer tumor tissues are loaded inside nanoparticles for the prevention of liver cancer: This example uses mouse liver cancer as a cancer model to illustrate how to prepare nanoparticles loaded with whole cell components of lung cancer and liver cancer tumor tissues. vaccine, and use the vaccine to prevent liver cancer. Lung cancer and liver cancer tumor tissues were first lysed to prepare water-soluble and water-insoluble fractions of the whole cell fraction and mixed at a ratio of 1:2. Then, using PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant, the nanovaccine loaded with water-soluble components and water-insoluble components of lung cancer tumor mass and liver cancer tumor mass was prepared by solvent evaporation method. This nanovaccine was then employed to prevent liver cancer.
（1）癌细胞的裂解及各组分的收集：方法同实施例3。(1) Lysis of cancer cells and collection of components: the method is the same as in Example 3.
（2）纳米疫苗的制备：本实施例中制备纳米疫苗采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，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。在制备疫苗时，水溶性组分为肺癌肿瘤组织水溶性组分和肝癌肿瘤组织水溶性组分的混合物，只分布于疫苗内部；非水溶性组分为肺癌肿瘤组织非水溶性组分和肝癌肿瘤组织非水溶性组分的混合物，只分布于疫苗内部。纳米粒子表面吸附免疫佐剂后所得纳米疫苗粒径为300nm左右，纳米粒子平均表面电位Zeta potential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且内外各半。空白纳米粒粒径为230nm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，空白纳米粒子表面吸附与纳米疫苗等量的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 nanoparticle preparation material PLGA molecular weight used is 24KDa-38KDa, and the immune adjuvant used is
poly(I:C) and poly(I:C) are both distributed inside the nanoparticles and adsorbed on the surface of the nanoparticles. When preparing the vaccine, the water-soluble component is a mixture of the water-soluble components of the lung cancer tumor tissue and the water-soluble components of the liver cancer tumor tissue, which are only distributed inside the vaccine; the water-insoluble components are the non-water-soluble components of the lung cancer tumor tissue and the A mixture of water-insoluble components of tumor tissue, distributed only inside the vaccine. The particle size of the nano-vaccine obtained after adsorption of the immune adjuvant on the surface of the nanoparticles is about 300nm, and the average surface potential Zeta potential of the nanoparticles 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, and the inside and outside are divided in half. The particle size of blank nanoparticles is about 230nm. Pure water containing poly (I:C) or 8M urea were used to replace the corresponding water-soluble components and insoluble components when preparing blank nanoparticles. The surface adsorption of blank nanoparticles and Equivalent poly(I:C) in nanovaccine.
（3）纳米疫苗用于癌症的预防：疫苗组和PBS对照组给药方案和肿瘤生长监测方案如实施例3。(3) Nano-vaccine for cancer prevention: the administration scheme and tumor growth monitoring scheme of the vaccine group and the PBS control group are as in Example 3.
（4）实验结果：如图32所示，与对照组相比，疫苗预防组肿瘤生长速度和小鼠生存期都有显著性差异。而且，疫苗组小鼠肿瘤接种后消失。而且，同时负载肺癌和肝癌肿瘤组织的疫苗预防效果好于只负载肝癌肿瘤组织的疫苗。由此可见，本发明所述的负载肺癌肿瘤组织和肝癌肿瘤组织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 32, compared with the control group, the tumor growth rate and the survival period of the mice in the vaccine prevention group were significantly different. Moreover, the tumors in the mice in the vaccine group disappeared after inoculation. Moreover, the preventive effect of the vaccine loaded with both lung cancer and liver cancer tumor tissues was better than that of the vaccine loaded only with liver cancer tumor tissues. It can be seen that the nano-vaccine loaded with water-soluble components and water-insoluble components in lung cancer tumor tissue and liver cancer tumor tissue of the present invention has a preventive effect on liver cancer.
实施例5 胰腺癌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胰腺癌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 Pancreatic cancer tumor tissue and colon cancer tumor tissue lysed components 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 pancreatic cancer 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Pan02胰腺癌肿瘤组织和MC38结肠癌肿瘤组织裂解组分按2:1的质量比例负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠胰腺癌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，水溶性组分为胰腺癌肿瘤组织水溶性组分和结肠癌肿瘤组织水溶性组分2:1的混合物；非水溶性组分为胰腺癌肿瘤组织非水溶性组分和结肠癌肿瘤组织非水溶性组分2:1的混合物。以PLGA（分子量7KDa-17KDa）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纳米疫苗。然后采用该纳米疫苗来治疗Pan02胰腺癌荷瘤小鼠体内的肿瘤。In this example, mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue lysate fractions were loaded on the inside and surface of nanoparticles at a mass ratio of 2:1 to prepare a nanovaccine. First, mouse pancreatic and colon cancer tumor tissues were obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea. When preparing the vaccine, the water-soluble component is a 2:1 mixture of the water-soluble component of pancreatic cancer tumor tissue and the water-soluble component of colon cancer tumor tissue; the water-insoluble component is the water-insoluble component of pancreatic cancer tumor tissue and the A 2:1 mixture of water-insoluble components of cancerous tumor tissue. Using PLGA (molecular weight 7KDa-17KDa) as the nanoparticle framework material and poly(I:C) as the immune adjuvant, the nanovaccine was prepared by the solvent evaporation method. The nanovaccine was then used to treat tumors in Pan02 pancreatic cancer-bearing mice.
（1）肿瘤组织的裂解及各组分的收集：在每只C57BL/6小鼠腋下皮下接种2×10
6个MC38结肠癌细胞或接种1×10
6个Pan02胰腺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000 mm
3时处死小鼠并摘取肿瘤组织。癌细胞的裂解方法及各组分的收集方法同上。
(1) Lysis of tumor tissue and collection of various components: Inoculate 2×10 6 MC38 colon cancer cells or 1×10 6 Pan02 pancreatic cancer cells subcutaneously in the armpit of each C57BL/6 mouse, and inoculate each mouse The mice were sacrificed when the inoculated tumors grew to a volume of about 1000 mm 3 , and the tumor tissues were harvested. The lysing method of cancer cells and the collection method of each component are the same as above.
（2）纳米疫苗的制备：本实施例中制备纳米疫苗制备方法同上。(2) Preparation of nano-vaccine: The preparation method of nano-vaccine in this example is the same as above.
（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400μL内部负载水溶性成分而表面负载原非水溶性成分的4mg 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，小鼠肿瘤监测和体积计算方法同上。
(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 400 μL of 4 mg PLGA nanoparticles loaded with water-soluble components inside and loaded with original water-insoluble components on the surface on the 4th day, 7th day, 10th day, 15th day and 20th day respectively. 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, mouse tumor monitoring and volume calculation methods were the same as above.
（4）实验结果：如图33所示，与对照组相比，纳米疫苗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，本发明所述的负载胰腺癌和结肠癌肿瘤组织全细胞组分的纳米疫苗对胰腺癌具有治疗效果。(4) Experimental results: As shown in Figure 33, compared with the control group, the tumor growth rate of the nano-vaccine 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 the whole cell components of pancreatic cancer and colon cancer tumor tissue according to the present invention has a therapeutic effect on pancreatic cancer.
实施例 6 乳腺癌和肺癌肿瘤组织全细胞组分负载于甘露糖修饰的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乳腺癌和肺癌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 breast cancer and lung cancer tumor tissues loaded inside mannose-modified microparticles for the prevention of lung cancer: This example uses mouse lung cancer as a cancer model to illustrate how to prepare breast cancer and lung cancer tumor tissues loaded A micron vaccine of whole cell components, and using 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，将小鼠乳腺癌和肺癌肿瘤组织裂解组分按1:4负载于微米粒子内部以制备微米疫苗。首先取得小鼠乳腺癌和肺癌的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 breast cancer and lung cancer tumor tissues were loaded inside the microparticles at a ratio of 1:4 to prepare micron vaccines. Tumor tissues of mouse breast and lung cancers were first obtained and lysed to prepare water-soluble fractions and original water-insoluble fractions dissolved in 8M urea. Then, using PLGA and mannose-modified PLGA as the microparticle framework material, and CpG as the immune adjuvant, the microvaccine was prepared by the solvent evaporation method. 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小鼠腋下皮下接种2×10
6个LLC肺癌细胞或者在每只BALB/c小鼠腋下接种2×10
6个4T1乳腺癌细胞，在小鼠所接种肿瘤长到1000
mm
3时处死小鼠并摘取肿瘤组织。其他处理方法同上。
(1) Lysis of tumor tissue and collection of various components: Inoculate 2×10 6 LLC lung cancer cells subcutaneously in the armpit of each C57BL/6 mouse or inoculate 2×10 6 in the armpit of each BALB/c mouse 4T1 breast cancer cells, the mice were sacrificed when the inoculated tumor grew to 1000 mm 3 and the tumor tissue was harvested. Other processing methods are the same as above.
（2）微米疫苗的制备：本实施例中制微米疫苗及作为对照的空微米粒采用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。未修饰PLGA，甘露糖修饰的PLGA的质量比为8: 2。微米疫苗内部负载癌细胞裂解物中水溶性成分而疫苗表面负载溶于8M尿素中原非水溶性成分，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1.30μm左右，平均表面电位为 -9 mV左右。每1 mg
PLGA微米粒子负载 65μg 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0.025mg。空白微米粒粒径为1.20 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 micro-vaccine: In this example, the micro-vaccine and the empty micro-particles used as a control adopt the double emulsion method in the solvent evaporation method, and the micro-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 is 8:2. The water-soluble components in the cancer cell lysate are loaded inside the micron vaccine, and the non-water-soluble components dissolved in 8M urea are loaded on the surface of the vaccine. The immune adjuvant used is CpG and CpG is distributed inside the micron particles. The preparation method is as mentioned above. The average particle size of the micron particles is about 1.30 μm, and the average surface potential is about -9 mV. per 1 mg
PLGA microparticles are loaded with 65 μg of protein or polypeptide components, and the CpG immune adjuvant used inside and outside of each 1 mg of PLGA microparticles is 0.025 mg. The particle size of the blank microparticles is about 1.20 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为模型小鼠制备肺癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天皮下注射400μL的4mg PLGA微米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL PBS。空白微米粒+裂解物对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL 空白微米粒和与疫苗所负载的等量的游离裂解物。在第0天给每只小鼠背部右下方皮下接种2×10
6个个LLC肺癌细胞。在实验中，小鼠肿瘤生长监测方法同上。
(3) Micro-vaccine targeting dendritic cells for cancer prevention: 6-8 week old female C57BL/6 were selected as model mice to prepare lung cancer tumor-bearing mice. The vaccine group was subcutaneously injected with 400 μL of 4 mg PLGA micron vaccine on days 35, 28, 21, 14 and 7 before tumor inoculation. 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 + lysate control group were subcutaneously injected with 400 μL of blank microparticles 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, 2×10 6 LLC lung cancer cells were subcutaneously inoculated into the lower right lower back of each mouse. In the experiment, the mouse tumor growth monitoring method was the same as above.
（4）实验结果：如图34所示，与PBS对照组和空白微米粒+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乳腺癌和肺癌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对肺癌具有预防效果。(4) Experimental results: As shown in Figure 34, compared with the PBS control group and the blank microparticle + 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 active targeting micron vaccine loaded with water-soluble components and non-water-soluble components in breast cancer and lung cancer tumor tissues has a preventive effect on lung cancer.
实施例7 肝癌和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制备负载有黑色素瘤和肝癌肿瘤全组织细胞组分的纳米疫苗并应用该疫苗预防肝癌。Example 7 The whole cell components of liver cancer and melanoma tumor tissue are loaded on the inside and surface of nanoparticles and the nano-vaccine with Bacillus Calmette-Guerin (BCG) as an immune adjuvant is used for the prevention of liver cancer: In this example, mouse liver cancer was used as a cancer model and Using BCG as an immune adjuvant to illustrate how to prepare nano-vaccine loaded with whole tissue cell components of melanoma and liver cancer tumors and apply the vaccine to prevent liver cancer.
本实施例中，首先裂解肝癌和黑色素瘤肿瘤组织的水溶性组分和非水溶性组分并分别按3:1混合。然后，以PLGA为纳米粒子骨架材料，以BCG为免疫佐剂采用溶剂挥发法制备纳米疫苗。In this example, the water-soluble and water-insoluble components of liver cancer and melanoma tumor tissues were firstly lysed and mixed at a ratio of 3:1. Then, using PLGA as the nanoparticle framework material and BCG as the immune adjuvant, the nanovaccine was prepared by the solvent evaporation method.
（1）肿瘤组织的裂解及各组分的收集：该实施例中肿瘤组织的裂解及裂解物收集同上。(1) Lysis of tumor tissue and collection of various components: Lysis of tumor tissue and collection of lysate in this embodiment are the same as above.
（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1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 components: The method of lysing BCG and collecting and solubilizing lysates in this example is the same as that of cancer cells in Example 1, 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内部和表面都负载癌细胞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天给每只小鼠背部右下方皮下接种2×10
6个Hepa1-6肝癌细胞。小鼠肿瘤生长监测方法同上。
(4) Nano-vaccine for the prevention of liver cancer: select female C57BL/6 as model mice to prepare liver cancer tumor-bearing mice. The vaccine group received subcutaneous injections of 200 μL of 2 mg PLGA nanoparticles loaded with water-soluble components in cancer cell lysate and 200 μL of 2 mg PLGA nanoparticles dissolved in 8M urea, the original water-insoluble components, were loaded on the surface. 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, 2×10 6 Hepa1-6 liver cancer cells were subcutaneously inoculated into the lower right back of each mouse. The mouse tumor growth monitoring method was the same as above.
（4）实验结果：如图35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，本发明所述负载肝癌和黑色素瘤肿瘤组织全细胞组分的纳米疫苗可以预防肝癌。(4) Experimental results: As shown in Figure 35, 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 liver cancer and melanoma tumor tissue in the present invention can prevent liver cancer.
实施例8 6M盐酸胍溶解乳腺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小鼠三阴性乳腺癌为癌症模型。首先对乳腺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为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织全细胞组分的微米疫苗。然后采用该微米疫苗来治疗4T1乳腺癌荷瘤小鼠体内的肿瘤。Example 8 6M guanidine hydrochloride dissolves tumor tissue components of breast cancer and colon cancer and loads them inside and on the surface of microparticles for the treatment of breast cancer: This example illustrates how to use 6M guanidine hydrochloride to dissolve whole cell components and prepare whole cell components loaded with microparticles Micro-vaccines of cellular components to treat breast cancer. In this example, 4T1 mouse triple-negative breast cancer was used as a cancer model. First, inactivate and denature the tumor tissue cells of breast cancer and colon cancer, lyse the tumor tissue with 6M guanidine hydrochloride and dissolve the whole cell components. Then, using PLGA as the microparticle framework material and CpG as the immune adjuvant, the micron vaccine loaded with the whole cell components of the tumor tissue was prepared by the solvent evaporation method. The microvaccine was then used to treat tumors in 4T1 breast cancer tumor-bearing mice.
（1）肿瘤组织的裂解及各组分的收集：在BALB/c小鼠右腋下皮下接种2×10
6个4T1细胞或者在C57BL/6小鼠右腋下皮下接种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。将肿瘤组织切块后加入胶原酶后研磨，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6M盐酸胍裂解乳腺癌和结肠癌肿瘤组织细胞并溶解组织裂解物，将乳腺癌的肿瘤组织裂解物与结肠癌肿瘤组织裂解物按照5:1比例混合后即为制备疫苗的原料来源。
(1) Lysis of tumor tissue and collection of various components: subcutaneously inoculate 2×10 6 4T1 cells in the right underarm of BALB/c mice or subcutaneously inoculate 2×10 6 MC38 in the right underarm of C57BL/6 mice For colon cancer cells, the mice were sacrificed when the tumor grew to a volume of 1000 mm 3 and the tumor tissue was harvested. The tumor tissue was cut into pieces, added with collagenase, and then ground, filtered through a cell strainer, and the filtered tumor tissue cells were collected. The obtained tumor tissue cells were inactivated and denatured respectively by ultraviolet light and high temperature heating, and then 6M guanidine hydrochloride was used to lyse the breast cancer and colon cancer tumor tissue cells and dissolve the tissue lysates, and the breast cancer tumor tissue lysates were combined with the colon cancer tumor tissue The lysate is mixed according to the ratio of 5:1, which is the raw material source for preparing the vaccine.
（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），的制备方法如前所述。采用CpG为免疫佐剂。所制备微米疫苗平均粒径为2.6μm左右，微米粒子表面Zeta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0.02mg且内外各半。无佐剂的微米疫苗性质与含CpG佐剂的疫苗相同，只是不含有CpG佐剂。(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 2.6 μm, and the Zeta potential on the surface 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 0.02 mg in total, and the inside and outside are divided into half. Unadjuvanted micron vaccines have the same properties as CpG-adjuvanted vaccines, except that they do not contain CpG adjuvant.
（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+游离裂解物对照组在第4天，第7天，第10天，第15天和第20天分别皮下注射等量肿瘤组织裂解物，以及负载有等量CpG而不负载任何裂解物成分的4mg
PLGA空白微米粒。在实验中，小鼠肿瘤体积监测和计算方法同上。
(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. The control group of blank microparticles + free lysate was subcutaneously injected with the same 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 lysate 4mg PLGA blank micron particles of the material composition. In the experiment, the mouse tumor volume was monitored and calculated in the same way as above.
（4）实验结果：如图36所示，与对照组相比，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。而且，含有佐剂的微米疫苗治疗效果好于无佐剂的微米疫苗。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 36, compared with the control group, the growth rate of the tumor in the micron vaccine administration group was significantly slower and the survival period of the mice was significantly prolonged. Moreover, the therapeutic effect of adjuvanted micron vaccines is better than that of non-adjuvanted micron vaccines. 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 黑色素瘤肿瘤组织和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黑色素瘤肿瘤组织和肺癌癌细胞的全细胞组分的纳米疫苗，并应用该疫苗预防黑色素瘤。 本实施例中，首先裂解B16F10黑色素瘤肿瘤组织和LLC癌细胞以制备相应的水溶性组分和溶于8M尿素的非水溶性组分。然后将来自肿瘤组织的水溶性组分和来自癌细胞的水溶性组分按质量比1∶1混合即为实验中使用的水溶性组分；将来自肿瘤组织的非水溶性组分和来自癌细胞的非水溶性组分按质量比1∶1混合即为实验中使用的非水溶性组分；然后，以PLGA为骨架材料，以胶体锰为免疫佐剂制备纳米疫苗。Example 9 Whole cell components of melanoma tumor tissue and lung cancer cancer cells loaded on the inside and surface of nanoparticles for the prevention of melanoma: This example illustrates how to prepare whole cell components loaded with melanoma tumor tissue and lung cancer cancer cells nano-vaccine and apply the vaccine to prevent melanoma. In this example, firstly, B16F10 melanoma tumor tissue and LLC cancer cells were lysed to prepare corresponding water-soluble fractions and water-insoluble fractions dissolved in 8M urea. Then the water-soluble components from tumor tissue and the water-soluble components from cancer cells were mixed at a mass ratio of 1:1 to be the water-soluble components used in the experiment; the water-insoluble components from tumor tissues and the water-soluble components from cancer cells The water-insoluble components of the cells are mixed according to the mass ratio of 1:1, which is the water-insoluble components used in the experiment; then, the nano-vaccine is prepared with PLGA as the skeleton material and colloidal manganese as the immune adjuvant.
（1）肿瘤组织及癌细胞的裂解及各组分的收集：B16F10黑色素瘤肿瘤组织和LLC肺癌癌细胞的裂解方法同上，在黑色素瘤肿瘤组织或LLC癌细胞裂解后，加入DNA降解酶作用20分钟，然后将裂解物以8000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分；用8M尿素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。各组分分别按照1:1的质量比例混合即为制备疫苗的原料来源。(1) Lysis of tumor tissue and cancer cells and collection of various components: The lysis method of B16F10 melanoma tumor tissue and LLC lung cancer cells is the same as above. Minutes, then centrifuge the lysate at a speed of 8000g for 5 minutes and take the supernatant, which is the water-soluble component soluble in pure water; dissolve the precipitate with 8M urea to remove the non-water-soluble component that is insoluble in pure water Converted to soluble in 8M urea aqueous solution. Each component is mixed according to the mass ratio of 1:1, which is the raw material source for preparing the vaccine.
（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25 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520nm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190μg蛋白质或多肽组分。空白纳米粒粒径为470nm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 25 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 allowed to react at room temperature for 10 min before use. The average particle size of the nanovaccine loaded with whole cell components is about 520nm, and the surface potential of the nanovaccine is about -5mV; each 1 mg PLGA nanoparticle is loaded with about 190μg protein or polypeptide component. The particle size of the blank nanoparticles is about 470nm, and the blank nanoparticles are loaded with the same 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37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，负载黑色素瘤肿瘤组织和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 37, 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 melanoma tumor tissue and lung cancer cells has a preventive effect on melanoma.
Claims (10)
- 一种基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，其特征在于，包括纳米和/或微米粒子、全细胞组分混合物；所述全细胞组分混合物为多种癌细胞和/或肿瘤组织全细胞组分。A nano and/or micro vaccine system for preventing or treating cancer based on a variety of cancer cells and/or tumor tissue whole cell components, characterized in that it includes nano and/or micro particles and a mixture of whole cell components; The whole cell component mixture is whole cell components of various cancer cells and/or tumor tissues.
- 根据权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，其特征在于，所述全细胞组分为不同癌细胞和/或不同肿瘤组织中全细胞的水溶性成分混合物和/或非水溶性成分混合物，所述水溶性成分为细胞或组织中的可溶于纯水或不含增溶剂的水溶液中的原水溶性部分，所述非水溶性成分为细胞或组织中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 nano and/or micro vaccine system for preventing or treating cancer based on multiple cancer cells and/or tumor tissue whole cell components according to claim 1, characterized in that, the whole cell components are different cancer cells and/or or a mixture of water-soluble components and/or a mixture of water-insoluble components of whole cells in different tumor tissues, the water-soluble components are the original water-soluble parts in cells or tissues that are soluble in pure water or aqueous solutions without solubilizers, The water-insoluble component is the original water-insoluble part of cells or tissues that is changed from being insoluble in pure water to being soluble in an aqueous solution containing a solubilizing agent or in an organic solvent through a solubilization method.
- 根据权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，其特征在于，所述全细胞组分混合物由两种或两种以上的癌细胞和/或肿瘤组织制备；所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的疫苗系统的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 nano and/or micro vaccine system for preventing or treating cancer based on multiple cancer cells and/or tumor tissue whole cell components according to claim 1, wherein the whole cell component mixture consists of two or two More than one cancer cell and/or tumor tissue preparation; the surface of the vaccine system for preventing or treating cancer based on the whole cell components of multiple cancer cells and/or tumor tissue may not be connected with a target head with active targeting function or Connect the target head with active targeting function.
- 根据权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，其特征在于，所述全细胞组分混合物被包载于纳米和/或微米粒子内部和/或负载于纳米和/或微米粒子表面；所述纳米和/或微米粒子内部和/或表面还包括免疫佐剂。The nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components according to claim 1, characterized in that, the whole cell component mixture is entrapped in nano And/or the interior of the micron particle and/or loaded on the surface of the nanometer and/or micron particle; the interior and/or surface of the nanometer and/or micron particle also include an immune adjuvant.
- 根据权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，其特征在于，纳米粒子为纳米级尺寸的粒子；微米粒子为微米级尺寸的粒子；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；所述纳米和/或微米粒子的形状为球形、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。According to the nano and/or micro vaccine system for preventing or treating cancer based on multiple cancer cells and/or tumor tissue whole cell components according to claim 1, it is characterized in that the nanoparticles are particles of nanoscale size; the micro particles are Particles of micron-scale size; the preparation materials of the nanometer and/or micrometer particles include organic synthetic polymer materials, natural polymer materials or inorganic materials; the shape of the nanometer and/or micrometer particles is spherical, ellipsoidal, barrel-shaped , polygon, rod, sheet, line, worm, square, triangle, butterfly or disc.
- 根据权利要求5所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统，其特征在于，所述纳米疫苗和纳米粒子的粒径分别为1nm-1000nm；所述微米疫苗和微米粒子的粒径分别为1μm-1000μm；所述纳米疫苗和/或微米疫苗表面为电中性，带负电或者带正电。According to the nano and/or micro vaccine system for preventing or treating cancer based on multiple cancer cells and/or tumor tissue whole cell components according to claim 5, it is characterized in that the particle diameters of the nano vaccine and the nano particle are respectively 1nm-1000nm; the particle size of the micro-vaccine and the micro-particles is 1 μm-1000 μm respectively; the surface of the nano-vaccine and/or the micro-vaccine is electrically neutral, negatively charged or positively charged.
- 根据权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米疫苗和/或微米疫苗系统，其特征在于，癌细胞或肿瘤组织中有一种与用于预防或治疗的癌症类型相同。The nano-vaccine and/or micro-vaccine system for the prevention or treatment of cancer based on multiple cancer cells and/or tumor tissue whole cell components according to claim 1, characterized in that, there is one in cancer cells or tumor tissue that is used for The same type of cancer is prevented or treated.
- 权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统的制备方法，其特征在于，将多种癌细胞和/或肿瘤组织全细胞组分负载于纳米和/或微米粒子内部和/或表面，得到所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统；或者将多种癌细胞和/或肿瘤组织全细胞组分、免疫佐剂负载于纳米和/或微米粒子内部和/或表面，得到所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统。The preparation method of the nano and/or micro vaccine system for the prevention or treatment of cancer based on the whole cell components of multiple cancer cells and/or tumor tissues according to claim 1, characterized in that, multiple cancer cells and/or tumor tissues Whole cell components are loaded inside and/or on the surface of nano and/or micro particles to obtain the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components; or loading multiple cancer cells and/or tumor tissue whole cell components and immune adjuvants inside and/or on the surface of nano and/or micro particles to obtain the Nano and/or micro vaccine systems for the prevention or treatment of cancer.
- 权利要求1所述基于多种癌细胞和/或肿瘤组织全细胞组分的预防或治疗癌症的纳米和/或微米疫苗系统在制备预防和/或治疗癌症的药物中的应用。The application of the nano and/or micro vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components in claim 1 in the preparation of drugs for preventing and/or treating cancer.
- 根据权利要求9所述的应用，其特征在于，癌细胞或肿瘤组织中有一种与用于预防或治疗的癌症类型相同。The use according to claim 9, characterized in that one of the cancer cells or tumor tissue is the same type of cancer used for prevention or treatment.
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