Vaccine system for preventing or treating cancer based on one or more cancer cells and / or whole cell fractions of tumor tissue or mixtures thereof

Abstract

The present application belongs to the field of immunotherapy, and discloses a vaccine system for preventing or treating cancer based on one or more cancer cells and / or whole cell components of tumor tissue or a mixture thereof, and its preparation method and application, wherein modified nano-sized or micron-sized particles are used to deliver water-soluble components and / or water-insoluble components of whole cell components of one or more cancer cells and / or tissues, and are used to prepare vaccines for preventing and treating cancer. The water-soluble part and / or water-insoluble part are all loaded in nanoparticles or micron particles, that is, the mutated protein or polypeptide is all loaded in the nano-vaccine or micron vaccine. These immunogenic substances in the whole cell components or a mixture thereof can be used for the prevention and treatment of cancer. Modification treatments such as adding chemical modification and adding charged substances in the vaccine preparation process can increase the amount of antigens and make the vaccine more effective. Therefore, the vaccine system of the present invention can prevent and / or treat cancer.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy, and specifically relates to a nano or micro cancer vaccine based on one or more cancer cells and / or tumor tissues and its modification and processing preparation technology, and especially to a cancer vaccine based on the whole cell components of cancer cells and / or tumor tissues of one or more cancers and its application in the prevention and treatment of cancer. Background Art

[0002] Immunity is a physiological function of the human body. In recent years, immune technology has developed rapidly, especially in the field of cancer immunotherapy. It has been found that the human immune system and various immune cells play a key role in 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 methods for cancer immunotherapy and prevention. The basis for developing cancer vaccines is to select suitable cancer antigens to activate the human immune system to recognize abnormal mutated cancer cells, and cancer cells or cancer tumor tissues themselves are the best sources of cancer antigens. Scientists have used new technologies to analyze and identify cancer-specific or cancer-related antigenic peptides from tumor cells of cancer patients, but such methods are time-consuming, labor-intensive and costly. Moreover, the methods used are to extract and analyze the differences between cancer cells and normal cells from the water-soluble components of cancer cells and then find different polypeptides. Therefore, such methods and techniques can only find a limited number of antigenic polypeptides with good water solubility, which greatly limits the application of such methods. The inventors previously disclosed that the whole cell components of cancer cells or cancer tissues are used as the source of vaccines for the prevention and treatment of cancer. This is a promising method, but its application effect needs to be improved. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a modification method to improve the efficacy of a micro or nano vaccine based on a whole cell component or a mixture thereof loaded with one or more cancer cells and / or tumor tissues in the prevention or treatment of cancer. The present invention uses a modification method such as solidification treatment and the addition of positively charged substances in the preparation process of the nano or micro vaccine to increase the antigen loading of the vaccine, and thus improve the prevention and treatment efficacy of the vaccine.

[0004] To achieve the purpose of the present invention, the present invention adopts the following technical solution:

[0005] A nano and / or micro vaccine system for preventing or treating cancer based on one or more whole cell components of cancer cells and / or tumor tissues or a mixture thereof, comprising nano and / or micro particles, whole cell components or a mixture thereof, and a modifier for increasing antigen loading or immunogenicity; the whole cell components or a mixture thereof are one or more whole cell components of cancer cells and / or tumor tissues or a mixture thereof; the nano vaccine and / or micro vaccine uses appropriate modification technology during the preparation process to increase the antigen loading and / or immunogenicity of the vaccine and thereby improve the efficacy of the vaccine.

[0006] The vaccine system for preventing or treating cancer based on one or more whole cell components of cancer cells and / or tumor tissues or a mixture thereof of the present invention is a nano or micro vaccine system, called a nano vaccine or a micro vaccine, which can prevent or treat cancer and is composed of nano or micro particles and whole cell components or a mixture thereof, or a modified substance, or composed of nano or micro particles and whole cell components or a mixture thereof, an immune adjuvant, or a modified substance, which are carried by the particles; the whole cell components are a mixture of water-soluble components and / or water-insoluble components or a mixture thereof of whole cells in cancer cells and / or tumor tissues. The mixture may be, but is not limited to, a mixture of water-soluble components, or a mixture of water-insoluble components, or a mixture of all and / or part of water-soluble components with all and / or part of water-soluble components.

[0007] The vaccine system for preventing or treating cancer according to the present invention is based on whole cell components of one or more cancer cells and / or tumor tissues or a mixture thereof, wherein the whole cell components are obtained by whole cell lysis of one or more cancer cells and / or tumor tissues, or the whole cell components are obtained by whole cell lysis of one or more cancer cells and / or tumor tissues and then processed, or the whole cell components are obtained by whole cell processing of one or more cancer cells and / or tumor tissues and then lysis.

[0008] The method for preparing the vaccine system for preventing or treating cancer based on one or more cancer cells and / or tumor tissue whole cell components or their mixtures of the present invention is that, in the presence of a modifier, one or more cancer cells and / or tumor tissue whole cell components or their mixtures 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 one or more cancer cells and / or tumor tissue whole cell components; or in the presence of a modifier, one or more cancer cells and / or tumor tissue whole cell components or their mixtures and immune adjuvants are loaded inside and / or on the surface of nano- and / or micro-particles to obtain the vaccine system for preventing or treating cancer based on one or more cancer cells and / or tumor tissue whole cell components. In the preparation process of the nano- and / or micro-vaccine, the inside and / or the surface of the nano-particles can be modified once or multiple times to increase the amount of antigen loaded by the vaccine and / or the immunogenicity of the antigen and thereby improve the effect of the vaccine. Specifically, the vaccine system for preventing or treating cancer based on a variety of cancer cells and / or whole cell components of tumor tissues described in the present invention can be prepared according to existing preparation methods for nano-sized particles and micron-sized particles, including but not limited to common solvent evaporation methods, dialysis methods, extrusion methods, and hot melt methods, and modified substances are used during the preparation process to increase the antigen loading capacity. In some embodiments, the vaccine system is prepared by the emulsion method in the solvent evaporation method. In the preparation process of nano- or micron-based vaccines, one or more steps of solidification, addition of positively charged substances, and other treatments can be performed to increase the antigen loading capacity or increase the immunogenicity of the antigen and thereby improve the efficacy of the vaccine.

[0009] The active ingredient whole cell component or mixture of the vaccine system for preventing or treating cancer based on one or more cancer cells and / or tumor tissue whole cell components or mixtures thereof of the present invention is a mixture of water-soluble components of whole cells and / or water-insoluble components or mixtures thereof, prepared from one or more cancer cells and / or tumor tissues, and can be prepared from one or more cancer cells, one or more tumor tissues, or a mixture of more than one cancer cell and tumor tissue. In the preparation process, the loading amount of nano-vaccine or micro-vaccine antigens can be increased by using modification techniques such as solidification and / or adding positively charged substances, which can improve the efficacy of the vaccine, and this is the creativity of the present invention. The modification technology used in the preparation process to increase the antigen loading or immunogenicity, that is, the modification treatment of the inside and / or surface of the nanoparticles includes but is not limited to one or more steps of solidification, biomineralization, covalent modification, addition of positively charged substances, and addition of negatively charged substances; the above-mentioned modifiers for increasing the antigen loading or immunogenicity are derived from this, and the present invention embodies the modifiers for increasing the antigen loading or immunogenicity on the vaccine system through one or more methods of solidification, biomineralization, covalent modification, addition of positively charged substances, and addition of negatively charged substances; preferably, the modification is one or more of siliconization, calcification, addition of positively charged substances, and addition of negatively charged substances.

[0010] The vaccine system for preventing or treating cancer based on one or more cancer cells and / or tumor tissue whole cell fractions or mixtures thereof of the present invention is used to prevent or treat cancer and its recurrence. In the vaccine system for preventing or treating cancer based on one or more cancer cells and / or tumor tissue whole cell fractions or mixtures thereof of the present invention, one of the cancer cells or tumor tissues is the same as the type of cancer to be prevented or treated.

[0011] In the vaccine system for preventing or treating cancer based on one or more whole cell components of cancer cells and / or tumor tissues or a mixture thereof of the present invention, the loading method is that the water-soluble components and water-insoluble components of the whole cells are separately or simultaneously encapsulated in the interior of the particles, and / or separately or simultaneously loaded on the surface of the particles. The whole cell components or their mixtures are loaded on the surface of nano- and / or micro-particles in ways including but not limited to adsorption, covalent bonding, charge interaction, hydrophobic interaction, solidification, encapsulation, etc. Specifically, the loading method is that the water-soluble components and the water-insoluble components of the whole cell are separately or simultaneously encapsulated in the particles, and / or separately or simultaneously loaded on the surface of the particles, including but not limited to the water-soluble components being loaded in the particles and loaded on the surface of the particles at the same time, the water-insoluble components being loaded in the particles and loaded on the surface of the particles at the same time, the water-soluble components being loaded in the particles and the water-insoluble components being loaded on the surface of the particles, the water-insoluble components being loaded in the particles and the water-soluble components being loaded on the surface of the particles, the water-soluble components and the water-insoluble components being loaded in the particles and only the water-soluble components being loaded on the surface of the particles, the water-soluble components and the water-insoluble components being loaded in the particles and only the water-soluble components being loaded on the surface of the particles, the water-soluble components being loaded in the particles and the water-soluble components and the water-insoluble components being loaded on the surface of the particles, the water-soluble components being loaded in the particles and the water-soluble components and the water-insoluble components being loaded on the surface of the particles, the water-soluble components and the water-insoluble components being loaded in the particles and the water-soluble components and the water-insoluble components being loaded on the surface of the particles at the same time, the water-soluble components and the water-insoluble components being loaded in the particles and the water-soluble components and the water-insoluble components being loaded on the surface of the particles at the same time. The water-soluble components and / or water-insoluble components in the whole cell components loaded on the surface of the nanoparticles and / or microparticles are loaded in one or more layers. Preferably, when multiple layers are loaded, there are modifications between the layers, which are derived from the above-mentioned modification technology.

[0012] In the vaccine system for preventing or treating cancer based on one or more cancer cells and / or whole cell components of tumor tissue or a mixture thereof of the present invention, the nano- and / or micro-particles also include immune adjuvants inside and / or on their surfaces, and the addition method includes loading them inside the nano-particles or micro-particles, or loading them on the surface of the nano-particles or micro-particles, or loading them inside and on the surface of the nano-particles or micro-particles at the same time. The immune adjuvant includes but is not limited to immune adjuvants of microbial origin, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicine and at least one of other categories; the immune adjuvant includes but is not limited to pattern recognition receptor agonists, BCG (BCG), manganese-related adjuvants, BCG cell wall skeletons, BCG methanol extraction residues, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhanced reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn bovine liver active peptide, Miquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly At least one of the effective ingredients of ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvant, CAF01, ginseng, and astragalus. Those skilled in the art will appreciate that the immune adjuvant may also use other substances that can enhance the immune response.

[0013] The surface of the vaccine system for preventing or treating cancer based on one or more cancer cells and / or whole cell components of tumor tissue or a mixture thereof described in the present invention may not be connected to a target head with active targeting function or may be connected to a target head with active targeting function; the target head can lead the delivery system to target specific cells; the specific cells or tissues are one or more of dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, basophils, lymph nodes, thymus, spleen, and bone marrow.

[0014] In the vaccine system for preventing or treating cancer based on one or more whole cell components of cancer cells and / or tumor tissues or a mixture thereof of the present invention, the whole cell components can be divided into two parts according to their solubility in pure water or an aqueous solution without a solubilizer: a water-soluble component and a water-insoluble component. The water-soluble component is the original water-soluble part that is soluble in pure water or an aqueous solution without a solubilizer, and the water-insoluble component is the original water-insoluble part that is insoluble in pure water, and is converted from being insoluble in pure water or an aqueous solution without a solubilizer to being soluble in an aqueous solution containing a solubilizer or in an organic solvent by a solubilization method. Both the water-soluble part and the water-insoluble part in the whole cell component can be dissolved by a solubilizing aqueous solution or an organic solvent containing a solubilizing agent. The solubilizing agent is at least one of the solubilizing agents that can increase the solubility of proteins or polypeptides in aqueous solutions; and the organic solvent is an organic solvent that can dissolve proteins or polypeptides. The solubilizing agent includes, but is not limited to, urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, alkaline solution with a pH greater than 7, acidic solution with a pH less than 7, various protein degrading enzymes, albumin, lecithin, high concentration inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij™-35, Octaethylene glycolmonododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630. It will be appreciated by those skilled in the art that the water-insoluble component may also be made soluble in pure water by other methods that can solubilize protein and polypeptide fragments. The organic solvent includes, but is not limited to, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, and ethyl acetate. It will be appreciated by those skilled in the art that the organic solvent may also be made soluble by other methods that can solubilize protein and polypeptide fragments by organic solvents.

[0015] In the vaccine system for preventing or treating cancer based on one or more cancer cells and / or whole cell components of tumor tissue or a mixture thereof of the present invention, the nano and / or micro particles are nano-sized particles and / or micro-sized particles. The particle size of the nano vaccine and nano-sized particles is 1nm-1000nm, preferably 50nm-800nm, and more preferably 100nm-600nm; the particle size of the micro vaccine and micro-sized particles is 1μm-1000μm, preferably 1μm-100μm, more preferably 1μm-10μm, and more preferably 1μm-5μm; the shape of the nano and / or micro particles is any common shape, including but not limited to spherical, ellipsoidal, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangle, butterfly-shaped or disc-shaped; the surface of the nano and / or micro vaccine system can be electrically neutral, negatively charged or positively charged.

[0016] In the vaccine system for preventing or treating cancer based on one or more cancer cells and / or whole cell components of tumor tissue or a mixture thereof according to the present invention, the preparation material of nano- and / or micron-particles is an organic synthetic polymer material, a natural polymer material or an 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 acid, synthetic polypeptide, and synthetic lipid. The natural polymer material is a biocompatible or degradable polymer material, including but not limited to lecithin, cholesterol, starch, lipids, sugars, polypeptides, sodium alginate, albumin, collagen, gelatin, and 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.

[0017] The vaccine system for preventing or treating cancer based on one or more whole cell components of cancer cells and / or tumor tissues or a mixture thereof of the present invention can deliver the loaded whole cell components to relevant immune cells, and activate and enhance the killing effect of the autoimmune system on cancer cells through the immunogenicity of the loaded components. Therefore, the present invention also provides the use of the vaccine system for preventing or treating cancer based on multiple whole cell components of cancer cells and / or tumor tissues in the preparation of vaccines for preventing and / or treating cancer.

[0018] The cancer vaccine system of the whole cell components or mixtures thereof described in the present invention can simultaneously use nanoparticles and / or microparticles that only load water-soluble components and nanoparticles and / or microparticles that only load water-insoluble components, use nanoparticles and / or microparticles that only load water-soluble components, use nanoparticles and / or microparticles that only load water-insoluble components, or use nanoparticles and / or microparticles that load both water-soluble components and water-insoluble components when preventing or treating diseases.

[0019] From the above technical scheme, it can be seen that the present invention provides a delivery system for delivering water-soluble components and / or water-insoluble components of cells using nano-sized or micron-sized particles, and its application in the preparation of vaccines for preventing and treating cancer. Because the whole cell components of the relevant cells or tissues are divided into two parts according to their solubility in pure water, a water-soluble part soluble in pure water and a water-insoluble part insoluble in pure water, and both the water-soluble part and the water-insoluble part are loaded in nanoparticles or micron particles, most of the mutated proteins or polypeptides produced by cancer in the cell components are loaded in nanoparticles or micron particles. The water-soluble part and the water-insoluble part in the cell components include the components of the entire cell; the water-soluble part and the water-insoluble part in the cell components can also be dissolved by an aqueous solution containing a solubilizing agent at the same time. Among them, the proteins, polypeptides and genes that are the same as normal cell components and have not mutated will not cause immune response because of the immune tolerance generated during the development of the autoimmune system; and the mutations of genes, proteins and polypeptides produced by cancer, etc., are immunogenic and can activate immune response because there is no immune tolerance generated during the development of the autoimmune system. The immunogenic substances produced by disease mutation in the whole cell components can be used for cancer treatment. The higher the antigen content in the nano-vaccine or micro-vaccine, the less the vaccine is needed when used, and the better the efficacy at the same dose. For this reason, the present invention proposes that the vaccine can increase the antigen loading or immunogenicity through solidification, biomineralization, addition of positively charged substances, addition of negatively charged substances, covalent modification and other methods, thereby enhancing the efficacy of the vaccine.

[0020] The cancer vaccine system of the present invention can be used to prepare vaccines for preventing and / or treating cancer. When used as a cancer vaccine to prevent and treat cancer, the vaccine of the present invention can be administered multiple times before or after cancer occurs or after surgical removal of tumor tissue to activate the body's immune system, thereby delaying the progression of cancer, treating cancer, or preventing the recurrence of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0022] Figure 1 Schematic diagram of the preparation process and application field of the vaccine of the present invention; a: schematic diagram of collecting water-soluble components and water-insoluble components separately and preparing nano-vaccines or micro-vaccines; b: schematic diagram of using a solubilizing solution containing a solubilizing agent to dissolve whole-cell components and prepare nano-vaccines or micro-vaccines; when encapsulating and loading components, modification technology is used to increase the loading capacity.

[0023] Figure 2 Schematic diagram of the structure of nano-sized particles or micron-sized particles carrying water-soluble and water-insoluble cellular components.

[0024] Figure 3 Schematic diagram of the structure of nano-sized particles or micron-sized particles carrying water-soluble and water-insoluble cellular components.

[0025] Figure 4 Schematic diagram of the structure of nanoparticles or microparticles modified with active targeting heads and loaded with water-soluble and water-insoluble cellular components.

[0026] Figure 5 Schematic diagram of the structure of nanoparticles or microparticles modified with active targeting heads and loaded with water-soluble and water-insoluble cellular components.

[0027] Figure 6 Schematic diagram of the structure of nanoparticles or microparticles modified with active targeting heads and loaded with water-soluble and water-insoluble cellular components.

[0028] Figure 7 The experimental results of mouse tumor growth rate and survival period in Example 1;

[0029] Figure 8 The experimental results of mouse tumor growth rate and survival period in Example 2;

[0030] Fig. 9 The experimental results of mouse tumor growth rate and survival period in Example 3;

[0031] Fig.10 The experimental results of mouse tumor growth rate and survival period in Example 4;

[0032] Fig.11 The experimental results of mouse tumor growth rate and survival period in Example 5;

[0033] Fig.12 The experimental results of mouse tumor growth rate and survival period in Example 6;

[0034] Fig.13 The results of the mouse tumor experiment in Example 7;

[0035] Fig.14 The experimental results of mouse tumor growth rate and survival period in Example 8;

[0036] Fig.15 The experimental results of mouse tumor growth rate and survival period in Example 9;

[0037] Fig.16 The experimental results of mouse tumor growth rate and survival period in Example 10;

[0038] Fig.17 The experimental results of mouse tumor growth rate and survival period in Example 11;

[0039] Figures 7 to 17 a, experimental results of tumor growth rate when nano- or micro-vaccines are used to prevent or treat cancer (n≥8); b, experimental results of mouse survival when nano- or micro-vaccines are used to 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, and the significant difference in Figure b was analyzed by Kaplan-Meier and log-rank test; ** indicates that the vaccine group was significantly different from the PBS blank control group at p<0.005; ## indicates that the vaccine group was significantly different from the blank nanoparticle + cell lysate control group at p<0.005; *** indicates that the vaccine group was significantly different from the PBS blank control group at p<0.0005; ### indicates that the vaccine group was significantly different from the blank nanoparticle + cell lysate control group at p<0.0005. $ indicates that there is a significant difference between the vaccine group and the unmodified vaccine group, p < 0.05; $$ indicates that there is a significant difference between the vaccine group and the unmodified vaccine group, p < 0.005; = ' represents the difference between the frozen silicification treated vaccine group and the room temperature silicification treated vaccine group (p < 0.05), which is significant; & represents the difference between the vaccine group modified by both low-temperature silicification and addition of charged substances and the vaccine group only modified by low-temperature silicification (p < 0.05), which is significant; δ represents the difference between the vaccine group treated by both low-temperature silicification and addition of charged substances and the vaccine group only treated by addition of charged substances (p < 0.05), which is significant; θ represents the difference between the calcification treated vaccine group and the calcification treated vaccine group (p < 0.05), which is significant. DETAILED DESCRIPTION

[0040] The present invention discloses a nanoscale or micron-scale broad-spectrum cancer vaccine system loaded with one or more cancer cells and / or whole cell components of tumor tissue or a mixture thereof and its application to prevent or treat cancer. In the process of vaccine preparation, the present invention increases the antigen loading amount and thereby improves the efficacy of the vaccine by methods such as biomineralization and addition of cationic substances. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and they are all deemed 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 modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0041] The present invention first obtains water-soluble components soluble in pure water or an aqueous solution without a solubilizer after lysing cancer cells and / or tissues, and then uses a solubilizing aqueous solution containing a solubilizer to dissolve water-insoluble components in the solubilizing solution, so that all cell components can be converted into components soluble in an aqueous solution and then loaded inside and outside nanoparticles or microparticles to prepare nano- or micro-vaccines for cancer prevention and treatment. In practical applications, the whole cell components can also be directly dissolved in a solubilizing aqueous solution containing a solubilizer after the cells or tissues are lysed without separately collecting the water-soluble components and the water-insoluble components, and the whole cell components dissolved in the solubilizing aqueous solution are used to prepare nano- or micro-vaccines.

[0042] The present invention uses an aqueous solution containing a solubilizer to convert the components in the cell that are insoluble in pure water or an aqueous solution without a solubilizer into components that are soluble in a specific solubilizing solution and can be used to prepare nanoparticles and micron particles, thereby improving the comprehensiveness and immunogenicity of the antigenic substances or components carried by the nanoparticles or micron particles. The whole cell components in cancer cells and / or tumor tissues are divided into a water-soluble part that can be dissolved in pure water or an aqueous solution without a solubilizer and a water-insoluble part that can be dissolved in an aqueous solution with a certain solubilizer, and the water-soluble part and the water-insoluble part are encapsulated in the nanoparticles or micron particles and loaded on their surfaces, thereby ensuring that most of the antigenic substances are loaded in the prepared vaccine. The water-soluble part and the water-insoluble part in the cell components include the components and components of the entire cell. Among them, the proteins, polypeptides and genes that are the same as the normal cell components and have not mutated will not cause immune response because of the immune tolerance generated during the development of the autoimmune system; and the mutations of genes, proteins and polypeptides caused by cancer have immunogenicity and can activate the body's immune response to cancer cells because there is no immune tolerance generated during the development of the autoimmune system. These cancer cell-specific immunogenic substances produced by disease mutations in whole cell components can be used for the prevention and treatment of cancer.

[0043] The delivery system of the whole cell components or the mixture thereof of the present invention can be used to prepare vaccines for preventing and / or treating cancer. The preparation process and application fields thereof are as follows: Figure 1 As shown. During the preparation, the water-soluble components and the water-insoluble components can be collected separately after the cells or tissues are lysed, and the nano-vaccine or micro-vaccine can be prepared separately; or the cells or tissues can be directly lysed by using a solubilizing solution containing a solubilizing agent, and the whole cell components can be dissolved to prepare the nano-vaccine or micro-vaccine. During the preparation process, biomineralization and cationic substances can be added inside or on the surface of the vaccine to increase the antigen loading under the same conditions and thus improve the efficacy of the vaccine.

[0044] The whole cell fraction or its mixture of the present invention is obtained by whole cell lysis of one or more cancer cells and / or tumor tissues, or the whole cell fraction is obtained by whole cell lysis of one or more bacteria and then processed, or the whole cell fraction is obtained by whole cell processing of one or more bacteria and then lysis. The whole cell fraction can be treated before or (and) after lysis, including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc., before preparation of nano- or micro-vaccines; or the nano- or micro-vaccines can be directly prepared without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment before or (and) after cell lysis. In some embodiments of the present invention, tumor tissue cells are inactivated or (and) denatured before lysis. In actual use, they can also be inactivated or (and) denatured after cell lysis, or they can be inactivated or (and) denatured both before and after cell lysis. In some embodiments of the present invention, the inactivation or (and) denaturation treatment methods before or after cell lysis are ultraviolet irradiation and high temperature heating. In actual use, treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, freeze drying, etc. can also be used. It can be understood by those skilled in the art that in actual application, the technicians can make appropriate adjustments according to the specific circumstances. The obtained broad-spectrum cancer vaccine system can be connected to the surface without a target head with active targeting function or with a target head with active targeting function.

[0045] The schematic diagram of the structure of the vaccine system for preventing or treating cancer based on whole cell components of multiple cancer cells and / or tumor tissues according to the present invention is as follows: Figure 2-Figure 6 For the sake of simplicity, the modified substances are not shown, which will not affect the understanding of those skilled in the art. In actual use, only one specific structure of nanoparticles and / or microparticles can be used, or two or more nanoparticles and / or microparticles with different structures can be used simultaneously. Figure 2-Figure 3Schematic diagram of the structure of nano-sized particles or micron-sized particles carrying water-soluble and water-insoluble cell components, wherein 1: water-soluble components in cell or tissue components; 2, water-insoluble components in cell or tissue components; 3, immune adjuvant; 4, nanoparticle or micron particle; 5: the core part in the nanoparticle; 2.a-5.i, the nanoparticle or micron particle contains immune adjuvant on both the surface and inside; 6.a-9.i, the immune adjuvant is only distributed inside the nanoparticle or micron particle; 10.a-13.i, the nanoparticle or micron particle contains immune adjuvant only on the outer surface; 14.a-17.i, the nanoparticle or micron particle has no immune adjuvant on both the inside and the outer surface; 2.ai, 6.ai, 10.ai and 14.ai, the water-soluble components in cell or tissue components carried by the nanoparticle or micron particle When the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles in 3.ai, 7.ai, 11.ai and 15.ai are distributed inside the nanoparticles or microparticles, no obvious core is formed; when the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a core is formed, and the core can be generated during the preparation process or formed by using polymers or inorganic salts; when the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles in 4.ai, 8.ai, 12.ai and 16.ai are distributed inside the nanoparticles or microparticles, multiple cores are formed, and the core can be generated during the preparation process or formed by using polymers or inorganic salts; 5.ai, 9.ai, 13.ai and 17.ai, the water-soluble components or water-insoluble components of the cell or tissue components encapsulated by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles and are located in the outer layer of the formed inner core; a: the water-soluble components encapsulated inside and loaded on the surface of the nanoparticles or microparticles are all the water-soluble components of the cancer cell or tumor tissue components; b: the water-insoluble components encapsulated inside and loaded on the surface of the nanoparticles or microparticles are all the water-insoluble components of the cancer cell or tumor tissue components; c: the water-insoluble components encapsulated inside the nanoparticles or microparticles are all the water-soluble components of the cancer cell or tumor tissue components, and the water-soluble components loaded on the surface are all the water-soluble components of the cancer cell or tumor tissue components; d: the water-soluble components encapsulated inside the nanoparticles or microparticles are all the water-soluble components of the cancer cell or tumor tissue components, and the water-insoluble components loaded on the surface are all the water-insoluble components of the cancer cell or tumor tissue components; e: the water-soluble components and water-insoluble components of the cell or tissue components are encapsulated inside the nanoparticles or microparticles at the same time, and the water-soluble components and water-insoluble components of the cell or tissue components are also loaded on the surface of the nanoparticles or microparticles at the same time; f: The water-soluble components and water-insoluble components of the cell or tissue components are simultaneously encapsulated in the nanoparticles or microparticles, while the surface of the nanoparticles or microparticles only carries the water-soluble components of the cell or tissue components; g: The water-soluble components and water-insoluble components of the cell or tissue components are simultaneously encapsulated in the nanoparticles or microparticles, while the surface of the nanoparticles or microparticles only carries the water-insoluble components of the cell or tissue components; h: The water-insoluble components of the cell or tissue components are only encapsulated in the nanoparticles or microparticles, while the surface of the nanoparticles or microparticles simultaneously carries the water-soluble components and water-insoluble components of the cell or tissue components; i: The water-soluble components of the cell or tissue components are only encapsulated in the nanoparticles or microparticles, while the surface of the nanoparticles or microparticles simultaneously carries the water-soluble components and water-insoluble components of the cell or tissue components. . Figure 4-Figure 6Shown is a schematic diagram of the structure of nanoparticles or microparticles loaded with water-soluble and water-insoluble cell components modified with active targeting heads, wherein 1: water-soluble components in cell or tissue components; 2: water-insoluble components in cell or tissue components; 3: immune adjuvant; 4: nanoparticles or microparticles; 5: the core part of the nanoparticle; 6: target heads that can target specific cells or tissues. 2.a-5.i The nanoparticles or microparticles contain immune adjuvants both on the surface and inside; 6.a-9.i The immune adjuvants are only distributed inside the nanoparticles or microparticles; 10.a-13.i The nanoparticles or microparticles contain immune adjuvants only on the outer surface; 14.a-17.i There are no immune adjuvants inside or outside the nanoparticles or microparticles; 18.a-19i Cell components and / or immune adjuvants are only distributed inside the nanoparticles or microparticles; 20.ai, 23.ai Cell components and / or immune adjuvants are only distributed outside the nanoparticles or microparticles; 21.a-22.i Cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively. Figure 4-5In 2.a-2.i, 6.a-6.i, 10.a-10.i and 14.a-14.i, the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles without forming an obvious core; in 3.a-3.i, 7.a-7.i, 11.a-11.i and 15.a-15.i, the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles without forming an obvious core; The water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles 4.a-4.i, 8.a-8.i, 12.a-12.i and 16.a-16.i are distributed in multiple core parts of the nanoparticles or microparticles; the cells or tissue components carried by the nanoparticles or microparticles 5.a-5.i, 9.a-9.i, 13.a-13.i and 17.a-17.i are distributed in multiple core parts of the nanoparticles or microparticles. or tissue components are distributed in the outer layer of the inner core formed by the nanoparticles or micron particles; a: the nanoparticles or micron particles are loaded with water-soluble components in the cancer cells or tumor tissue components and on the surface; b: the nanoparticles or micron particles are loaded with water-soluble components in the cancer cells or tumor tissue components and on the surface; c: the nanoparticles or micron particles are loaded with water-soluble components in the cancer cells or tumor tissue components and on the surface are loaded with water-soluble components in the cancer cells or tumor tissue components; d: the nanoparticles or micron particles are loaded with water-soluble components in the cancer cells or tumor tissue components and on the surface are loaded with water-soluble components in the cancer cells or tumor tissue components; e: the nanoparticles or micron particles are loaded with water-soluble components and water-soluble components in the cells or tissue components at the same time, and the nanoparticles or micron particles are also loaded with water-soluble components and water-soluble components in the cells or tissue components at the same time; f: The nanoparticles or micron particles simultaneously encapsulate both water-soluble and water-insoluble components of cell or tissue components, while the surface of the nanoparticles or micron particles only carries water-soluble components of cell or tissue components; g: The nanoparticles or micron particles simultaneously encapsulate both water-soluble and water-insoluble components of cell or tissue components, while the surface of the nanoparticles or micron particles only carries water-insoluble components of cell or tissue components; h: The nanoparticles or micron particles only encapsulate water-insoluble components of cell or tissue components, while the surface of the nanoparticles or micron particles simultaneously carries water-soluble and water-insoluble components of cell or tissue components; i: The nanoparticles or micron particles only encapsulate water-soluble components of cell or tissue components, while the surface of the nanoparticles or micron particles simultaneously carries water-soluble and water-insoluble components of cell or tissue components. In Figure 6In a, b and c, the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles without forming an obvious core; in d, e and f, the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles are distributed in a core part inside the nanoparticles or microparticles; in g, h and i, the water-soluble components or water-insoluble components of the cells or tissue components carried by the nanoparticles or microparticles are distributed in multiple core parts inside the nanoparticles or microparticles; in j, k and l, the water-soluble components or water-insoluble components of the cells or tissue components encapsulated by the nanoparticles or microparticles are distributed in the outer layer of the core formed inside the nanoparticles or microparticles; a, d, g The nanoparticles or microparticles in and j are loaded with water-soluble components in cancer cells or tumor tissue components; the nanoparticles or microparticles in b, e, h and k are loaded with water-insoluble components in cancer cells or tumor tissue components; the nanoparticles or microparticles in c, f, i and l are loaded with both water-soluble components and water-insoluble components in cell or tissue components.

[0046] In an embodiment, the immune adjuvant is encapsulated in the nanoparticles or microparticles and loaded on the surface of the nanoparticles or microparticles at the same time. In actual use, the immune adjuvant can also be only encapsulated in the nanoparticles or microparticles, or only loaded on the surface of the nanoparticles or microparticles, or no immune adjuvant is added. In some embodiments, the present invention first encapsulates the water-soluble part or (and) water-insoluble part of the cell component that is soluble in pure water with a solubilizer and then loads it in the nanoparticles or microparticles, and loads the immune adjuvant at the same time; then, the water-soluble part or (and) water-insoluble part of the cell component is loaded on the surface of the nanoparticles, and the immune adjuvant is loaded at the same time. In this way, the loading capacity of the water-soluble component or water-insoluble component of the cell in the nanoparticles or microparticles can be maximized. In practical applications, it is also possible to directly use a solubilizing solution (such as an 8M urea aqueous solution or a 6M guanidine hydrochloride aqueous solution) containing a solubilizing agent to directly lyse cells or tissues and directly dissolve the whole cell component, and then prepare a nano vaccine or a micro vaccine.

[0047] The method for preparing nano vaccines and micro vaccines described in the present invention is a common preparation method. In some embodiments, the preparation of nano vaccines adopts the emulsion method in the solvent evaporation method, the nanoparticle preparation material used is an organic polymer polylactic acid-glycolic acid copolymer (PLGA) with a molecular weight of 24KDa-38KDa, 38KDa-54KDa or 7-17KDa, and the immune adjuvant used is poly (I: C), BCG (BCG) cGAS-STING agonist or CpG. Those skilled in the art can understand that in the actual application process, the technicians can make appropriate adjustments to the preparation method, preparation process, nanoparticle preparation materials used, the type and concentration of immune adjuvants, etc. according to the specific circumstances.

[0048] In the modification of the nanoparticle preparation process, the biomineralization method of the present invention uses the silicification and calcification methods. In practical applications, other methods that can increase the antigen loading amount, such as magnesium, can also be used.

[0049] In some embodiments, the specific preparation method of the double emulsion method used in the present invention is as follows:

[0050] Step 1: adding a first predetermined volume of an aqueous solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of a medical polymer material.

[0051] In some embodiments, the aqueous solution may contain various components in the cancer cell lysate and immune adjuvants poly(I:C), manganese adjuvant, BCG or CpG; the various components in the cancer cell lysate are water-soluble components or original water-insoluble components dissolved in 8M urea during preparation. The concentration of water-soluble components from cancer cells contained in the aqueous solution or the concentration of original water-insoluble components from cancer cells dissolved in 8M urea, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 1 ng / mL, which can load enough cancer antigens to activate related immune responses. The concentration of the immune adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0052] In some embodiments, the aqueous solution contains components of tumor tissue lysate and immune adjuvants poly(I:C), manganese adjuvant, BCG or CpG; each component of tumor tissue lysate is a water-soluble component or a originally water-insoluble component dissolved in 8M urea during preparation. The concentration of water-soluble components from tumor tissue contained in the aqueous solution or the concentration of originally water-insoluble components from tumor tissue dissolved in 8M urea, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 1 ng / mL, which can load enough cancer antigens to activate related immune responses. The concentration of the immune adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0053] 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 second predetermined concentration of the medical polymer material ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0054] In the present invention, PLGA or modified PLGA is selected because the material is a biodegradable material and has been approved by the FDA for use as a medical dressing. Studies have shown that PLGA has certain immunomodulatory functions and is therefore suitable as an adjuvant for vaccine preparation.

[0055] In practice, the second predetermined volume of the organic phase is set according to the ratio of the second predetermined volume of the organic phase to 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 is in the range of 1:1.1-1:5000, preferably 1:10. In 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 needed to adjust the size of the prepared nanoparticles or microparticles.

[0056] Preferably, when the aqueous phase solution is a lysate component solution, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL; when the aqueous phase solution is a lysate component / immune adjuvant solution, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. 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 to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion liquid is PBS buffer or physiological saline or pure water.

[0057] Step 2, subjecting the mixed solution obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute. Preferably, when stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, and the stirring time is greater than 1 minute, such as 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, and the time is greater than 0.1 second, such as 2 to 200 seconds; when homogenizing, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure when using a high pressure / ultra-high pressure homogenizer is greater than 5 psi, such as 20psi to 100psi, and the speed when using a high shear homogenizer is greater than 100 rpm, such as 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min-100 mL / min. Ultrasound, stirring, homogenization or microfluidics treatment is used for nano- and / or micron-size. The length of ultrasound time, stirring speed or homogenization pressure and time can control the size of the prepared nano- and / or micron-particles. Too large or too small a particle size will lead to a change in particle size.

[0058] Step 3, adding the mixture obtained after the treatment in step 2 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and performing an ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing a homogenization treatment or a microfluidic treatment. In this step, the mixture obtained in step 2 is added to the emulsifier aqueous solution and continues to be ultrasonicated or stirred for nano- or micronization. This step is for nano- or micronization. The length of the ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles or micron particles. Too long or too short will bring about changes in the particle size. For this reason, it is necessary to select a suitable ultrasonic time. In the present invention, the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds, the stirring speed is greater than 50rpm, such as 50 rpm to 500 rpm, and the stirring time is greater than 1 minute, such as 60 to 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, such as the stirring speed is 50 rpm to 1500 rpm, and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W, and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and when a high pressure / ultra-high pressure homogenizer is used, the pressure is greater than 20psi, such as 20psi to 100psi, and when a high shear homogenizer is used, the speed is greater than 1000 rpm, such as 1000 rpm to 5000rpm; when microfluidic treatment is used, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min-100 mL / min. Ultrasound, stirring, homogenization or microfluidics are used for nano- or micron-size. The length of ultrasound time, stirring speed or homogenization pressure and time can control the size of the prepared nano- or micron-particles. Too large or too small will lead to changes in particle size.

[0059] In the present invention, the emulsifier aqueous solution is a 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 set to 1:1.1-1:1000, preferably 2:5. In the specific implementation process, in order to control the size of nanoparticles or micron particles, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution and the concentration of this step are all based on the values ​​of obtaining nanoparticles or micron particles of appropriate size.

[0060] Step 4, adding the liquid obtained after the treatment in step 3 to a fourth predetermined volume of an aqueous emulsifier solution of a fourth predetermined concentration, and stirring until a predetermined stirring condition is met, or directly performing subsequent treatment without stirring.

[0061] In this step, the emulsifier aqueous solution is still PVA.

[0062] The fourth predetermined concentration is 5 mg / mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles or micron particles of suitable size. The selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. 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 the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or micron particles.

[0063] In the present invention, the predetermined stirring condition of this step is that the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized. Subsequent tests are not performed without stirring.

[0064] 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 re-suspend the remaining precipitate in a fifth predetermined volume of a solution of a fifth predetermined concentration containing water-soluble and / or water-insoluble components in the whole cell component, or re-suspend the remaining precipitate in a fifth predetermined volume of a solution of a fifth predetermined concentration containing water-soluble and / or water-insoluble components in the whole cell component and an adjuvant.

[0065] Step 6, after the mixed solution that meets the predetermined stirring conditions in step 5 is centrifuged at a speed greater than 100 RPM for more than 1 minute, the supernatant is removed, and the remaining precipitate is re-suspended in the sixth predetermined volume of solidification treatment reagent or biomineralization treatment reagent, centrifuged and washed after a certain period of time, and then the seventh predetermined volume of positively or negatively charged substances is added and allowed to react for a certain period of time.

[0066] In some embodiments of the present invention, the precipitate obtained in step 6 may not need to be freeze-dried after being resuspended in the seventh predetermined volume of charged substance, and subsequent experiments related to loading cancer cell / tissue lysate on the surface of nanoparticles or microparticles may be directly performed.

[0067] In some embodiments of the present invention, the precipitate obtained in step 6 is resuspended in an aqueous solution containing a drying protective agent and then vacuum dried at room temperature or freeze-vacuum dried. After drying, the cancer cell lysate is adsorbed on the surface of the nanoparticles or microparticles.

[0068] In the present invention, the freeze-drying protective agent is selected from trehalose or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the freeze-drying protective agent in this step is 2-6% by mass, such as 3-5%, and optimally 4%. The reason for this setting is to not affect the drying effect in the subsequent drying.

[0069] Step 7, drying the suspension containing the drying protective agent obtained in step 6, and keeping the dried material for later use.

[0070] Step 8, the suspension containing nanoparticles obtained in step 6 and resuspended in PBS (or physiological saline) with an eighth predetermined volume, or the dried substance containing nanoparticles or micron particles and a drying protective agent obtained in step 7 and resuspended in an eighth predetermined volume of PBS (or physiological saline), is mixed with the ninth predetermined volume of the water-soluble component or the water-insoluble component to obtain the nanovaccine or micron vaccine.

[0071] In the present invention, the modification and antigen loading steps of step 5 to step 8 can be repeated multiple times to increase the antigen loading amount. Moreover, when adding positively or negatively charged substances, substances with the same charge can be added multiple times or substances with different charges can be added alternately.

[0072] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble components or the original water-insoluble components in the cancer cell lysate or tumor tissue lysate is 0.1-100 mL. In actual use, the volumes and ratios of the two can be adjusted as needed.

[0073] In the present invention, the water-soluble component or the original water-insoluble component of the cancer cell lysate or tumor tissue lysate contains poly(I:C), manganese adjuvant, BCG (BCG) or CpG, and the concentration of poly(I:C), manganese adjuvant, BCG or CpG is greater than 1 ng / mL.

[0074] The particle size of the nano vaccine or micro vaccine is nanometer or micrometer, which can ensure that the vaccine is phagocytosed 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, and most preferably, the particle size is 100nm-600nm; the particle size of the micro vaccine is 1μm-1000μm, more preferably, the particle size is 1μm-100μm, more preferably, the particle size is 1μm-10μm, and most preferably, the particle size is 1μm-5μm. In this embodiment, the particle size of the nanoparticle vaccine is 100nm-600nm, and the particle size of the micro vaccine is 1μm-5μm.

[0075] In addition, in the present invention, urea and guanidine hydrochloride are used to solubilize the original water-insoluble components in the cancer cell lysate or tumor tissue lysate. In actual use, any other solubilizing substances that can dissolve the original water-insoluble components in the cancer cell lysate or tumor tissue lysate in an aqueous solution may also be used, such as sodium deoxycholate, SDS, alkaline solutions with a pH greater than 7, acidic solutions with a 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™-35, Octaethylene glycolmonododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630; or the above-mentioned solubilizing agents may be used to dissolve the water-soluble components and the water-insoluble components at the same time.

[0076] In addition, in the present invention, 8M urea and 6M guanidine hydrochloride aqueous solutions are used to solubilize the original water-insoluble components in the cancer cell lysate or tumor tissue lysate. In actual use, any other urea concentration or guanidine hydrochloride concentration that can dissolve the original water-insoluble components in the cancer cell lysate or tumor tissue lysate in an aqueous solution can also be used; or a solution containing a solubilizing agent can be used to simultaneously dissolve the water-soluble components and the water-insoluble components in the whole cell components of the cancer cells and / or tumor tissues.

[0077] In addition, in the present invention, the preparation of nano- and micro-vaccines adopts the double emulsion method supplemented by appropriate modification treatment, and any other method for preparing nano-particles or micro-particles may also be adopted in practice; the preparation material of nano- and micro-vaccines is PLGA, and any other material that can be used to prepare nano- or micro-particles may also be adopted in practice.

[0078] In addition, in the present invention, the water-soluble components in the cancer cell lysate or tumor tissue lysate or the original water-insoluble components dissolved in the solubilizing agent are respectively encapsulated in the nanoparticles and loaded on the surface of the nanoparticles. In actual use, the water-soluble components and the water-insoluble components in the cancer cell lysate or tumor tissue lysate can also be mixed and then encapsulated in the nanoparticles or loaded on the surface of the nanoparticles; or a solution containing a solubilizing agent can be used to simultaneously dissolve the water-soluble components and the water-insoluble components and then encapsulated in the nanoparticles or micron particles and / or loaded on the surface of the nanoparticles or micron particles.

[0079] In addition, in the present invention, poly(I:C), manganese adjuvant, BCG (BCG) and CpG are used as immune adjuvants. In practice, no immune adjuvant may be added or any other immune adjuvant with immune enhancement function may be added, such as pattern recognition receptor agonist, BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immune-enhancing reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn bovine liver active peptide, Miquimod, polysaccharide, curcumin, poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, double-stranded RNA, double-stranded DNA, aluminum hydroxide, CAF01, ginseng, astragalus and other effective ingredients of traditional Chinese medicine.

[0080] In addition, in the present invention, the vaccine used in some embodiments is a nano vaccine, and in some embodiments is a micro vaccine. Those skilled in the art can choose to use a nano vaccine and / or a micro vaccine according to actual conditions.

[0081] 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 combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0082] Unless otherwise specified, the methods used in the embodiments of the present invention are all conventional methods; the materials, reagents, etc. used can be obtained from commercial sources. The nano-sized particles or micron-sized particle structures, preparation methods, and strategies for use in disease treatment involved in the embodiments of the present invention are only representative methods. Other nano-sized particles or micron-sized particle structures, preparation methods, strategies for use in disease prevention or treatment, and strategies for combination with other drugs can also adopt the methods described in the present invention. The embodiments only list the application of the present invention in some cancers, but the present invention can also be used for any other type of cancer. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional replacement choices based on the existing technology on the basis of the technical ideas of the present invention, and are not limited to the specific records of the embodiments of the present invention. In actual application, the specific administration time, number of administrations, administration regimen, and combination with other drugs can be adjusted according to the situation.

[0083] Example 1 Whole cell components of melanoma tumor tissue loaded inside and on the surface of nanoparticles for the prevention of melanoma

[0084] This embodiment uses mouse melanoma as a cancer model to illustrate how to prepare a nano vaccine loaded with whole cell components of melanoma tumor tissue, and the vaccine is used to prevent melanoma. In this embodiment, B16F10 melanoma tumor tissue is first cracked to prepare water-soluble components and water-insoluble components of tumor tissue. Then, organic polymer material PLGA is used as a nanoparticle skeleton material, Polyinosinic-polycytidylic acid (poly(I:C)) is used as an immune adjuvant and a solvent volatilization method is used to prepare a nano vaccine loaded with water-soluble components and water-insoluble components of tumor tissue. In this embodiment, the method of siliconization and adding cationic substances is used to increase the load of antigens, and only one round of low-temperature siliconization treatment is performed.

[0085] (1) Lysis of tumor tissue and collection of various components

[0086] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16-F10 cells, and when the tumors grow to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were cut into pieces and ground, and pure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution. The above is the source of antigen raw materials for preparing vaccines.

[0087] (2) Preparation of Nanovaccines

[0088] Nano vaccine and the blank nanoparticles used as a control in the present embodiment are prepared by the multiple emulsion method in the solvent evaporation method, and in the nano vaccine preparation process, low temperature siliconization technology and two modification methods of adding charged substances are adopted to improve the antigen loading capacity. The nano vaccine of water-soluble components in the load full cell component and the nano vaccine of water-insoluble components in the load full cell component are prepared respectively when preparing, and then used together when using. The nano particle preparation material PLGA molecular weight adopted is 24KDa-38KDa, and the immunoadjuvant adopted is poly (I: C) and poly (I: C) both distributed in the nano particle inside and also loaded on the nano particle surface. The preparation method is as described above. In the preparation process, the antigen is first loaded inside the nanoparticles by the emulsion method. After the antigen (lysis component) is loaded inside, 100 mg of nanoparticles are centrifuged at 10000g for 20 minutes, and then the nanoparticles are resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), and then centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 10 min, and then fixed at -80℃ for 24 h, and then washed by centrifugation with ultrapure water, and then resuspended with 3 mL of PBS containing protamine (5 mg / mL) and polylysine (10 mg / mL) and acted for 10 min, and then washed by centrifugation at 10000g for 20 min, and resuspended with 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 min, Then, after centrifugation at 10000g for 20 minutes, the supernatant was removed and the precipitate was resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours; before the vaccine was injected, it was resuspended in 7 mL of PBS and then 3 mL of cancer tissue lysate containing immune adjuvant (protein concentration 50 mg / mL) was added and reacted at room temperature for 10 minutes to obtain a nanovaccine modified by frozen siliconization and addition of cationic substances with both internal and external lysates loaded. The average particle size of the nanovaccine is about 270nm, and the surface potential of the nanovaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 280μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg, and half is inside and half is outside.

[0089] The preparation method and steps of the unmodified nanovaccine are basically the same as those of the modified nanovaccine, except that the low-temperature siliconization and charged substance addition steps are not performed. In the preparation process, the antigen is first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysate component) is loaded inside, it is centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. Before the vaccine is used, it is resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate component containing adjuvant (protein concentration 50 mg / mL) is added and reacted at room temperature for 10 minutes to obtain a nanovaccine loaded with lysate inside and outside. The average particle size of the nanovaccine is about 230nm, and the surface potential of the nanovaccine is about -5mV; each 1mg PLGA nanoparticle is loaded with about 180μg of protein or polypeptide components, and the poly (I:C) immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.02mg, and half is inside and half is outside.

[0090] The particle size of blank nanoparticles is about 230 nm. When preparing blank nanoparticles, pure water or 8 M urea containing an equal amount of poly(I:C) is used to replace the corresponding water-soluble components and water-insoluble components.

[0091] (3) Nanovaccines for cancer prevention

[0092] The control groups of this study were PBS group, blank nanoparticles + cell lysate group and unmodified nanovaccine group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.

[0093] The dosing schedule for the nanovaccine group was as follows: 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components in the lysate and 200 μL of 2 mg PLGA nanovaccine loaded with water-insoluble components in 8 M urea were subcutaneously injected on days 42, 35, 28, 21, and 14 before melanoma inoculation. On day 0, 1.5 × 10 5 B16F10 cells.

[0094] The PBS control group was subcutaneously injected with 400 μL PBS on days 42, 35, 28, 21, and 14 before melanoma inoculation. On day 0, 1.5×10 5B16F10 cells. Blank nanoparticles + free lysate control group: 400 μL of blank nanoparticles and free lysate equal to the vaccine load were subcutaneously injected on days 42, 35, 28, 21, and 14 before melanoma inoculation. Blank nanoparticles and free lysate were injected at different sites. On day 0, 1.5×10 5 B16F10 cells.

[0095] In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0096] (4) Experimental results

[0097] like Figure 7 As shown, the tumors of mice in the vaccine group treated with the modified B16F10 tumor tissue whole cell component loaded during the nano vaccine preparation process disappeared completely after vaccination; while the tumors of mice in the vaccine group treated with the unmodified B16F10 tumor tissue whole cell component loaded disappeared 70% after vaccination; while the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger. In summary, the nano vaccine modified by siliconization and addition of cationic substances in the present invention has a good preventive effect on melanoma, and because more antigens can be loaded after modification, its preventive effect is better than the nano vaccine group that has not been modified during the preparation process.

[0098] Example 2 Whole cell components of melanoma cancer cells loaded inside and on the surface of nanoparticles for the prevention of melanoma

[0099] The present embodiment is to illustrate how to prepare the nano vaccine of the whole cell component of melanoma cancer cell load with mouse melanoma as cancer model, and use this vaccine to prevent melanoma.In the present embodiment, first cracking B16F10 melanoma cancer cell is to prepare water-soluble component and water-insoluble component.Then, take organic macromolecular material PLGA as nanoparticle skeleton material, take CpG as immunoadjuvant adopting solvent evaporation method to prepare the nano vaccine of the water-soluble component and water-insoluble component of cancer cell load.In the present embodiment, adopted siliconization, the method for adding cationic substance and anionic substance to increase the load capacity of antigen, and carried out two rounds of siliconization.

[0100] (1) Lysis of cancer cells and collection of components

[0101] The cultured B16F10 melanoma cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cells were resuspended in ultrapure water and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed of 3000g for 6 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into soluble in 8M urea aqueous solution. The above is the source of antigen raw materials for preparing vaccines.

[0102] (2) Preparation of Nanovaccines

[0103] Nano vaccine and the blank nanoparticle in contrast adopt the multiple emulsion method preparation in the solvent evaporation method in the present embodiment, adopt low temperature siliconization technology and two kinds of modification methods of adding charged substances to improve the antigenic load in the nano vaccine preparation process. Nano vaccine of water-soluble components in the load full cell component and the nano vaccine of water-insoluble components in the load full cell component are prepared respectively when preparing, and then used together when using. The nano particle preparation material PLGA molecular weight adopted is 24KDa-38KDa, and the immunoadjuvant adopted is that CPG and CpG are both distributed in the nano particle inside and are also loaded on the nano particle surface. The preparation method is as described above. In the preparation process, the antigen is first loaded inside the nanoparticles by the emulsion method. After the antigen (lysis component) is loaded inside, 100 mg of nanoparticles are centrifuged at 10000g for 20 minutes, and then the nanoparticles are resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), and then centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of silicate aqueous solution (containing 120 mM NaCl, 100 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 30 min, and then fixed at -80℃ for 48 h, and washed by centrifugation with ultrapure water, and then resuspended with 3 mL of PBS containing polyaspartic acid (10 mg / mL) and acted for 10 min, and then washed by centrifugation at 12000g for 18 min, and resuspended with 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 min, The cells were then centrifuged at 10,000 g for 20 minutes. Then, 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0) was used and fixed at room temperature for 10 min, then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, resuspended with 3 mL of PBS containing protamine (5 mg / mL) and polylysine (10 mg / mL) and acted for 10 min, then washed by centrifugation at 10,000 g for 20 min, resuspended with 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 min, then centrifuged at 10,000 g for 20 minutes, resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h; before vaccine injection, it was resuspended with 7 mL of PBS and then 3 mL of cancer cell lysate component containing adjuvant (protein concentration 50 mg / mL) was added and acted at room temperature for 10 min to obtain a nanovaccine modified by frozen siliconization and addition of cationic substances with lysate loaded both inside and outside.The average particle size of the nano vaccine is about 350nm, and the surface potential of the nano vaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 350μg of protein or polypeptide components, and the CpG immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, with half inside and half outside.

[0104] The preparation method and steps of the unmodified nanovaccine are basically the same as those of the modified nanovaccine, except that the steps of siliconization, addition of cationic substances and anionic substances are not performed. In the preparation process, the antigen is first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysate component) is loaded inside, it is centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. Before the vaccine is used, it is resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate component containing adjuvant (protein concentration 50 mg / mL) is added and reacted at room temperature for 10 minutes to obtain a nanovaccine loaded with lysate inside and outside. The average particle size of the nanovaccine is about 320nm, and the surface potential of the nanovaccine is about -5mV; each 1mg PLGA nanoparticle is loaded with about 180μg of protein or polypeptide components, and the CpG immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.02mg, and half is inside and half is outside.

[0105] The particle size of the blank nanoparticles is about 300 nm. When preparing the blank nanoparticles, pure water or 8M urea containing an equal amount of CpG is used to replace the corresponding water-soluble components and water-insoluble components.

[0106] (3) Nanovaccines for cancer prevention

[0107] The control groups of this study were PBS group, blank nanoparticles + cell lysate group and unmodified nanovaccine group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma tumor-bearing mice.

[0108] The dosing schedule for the nanovaccine group was as follows: 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components in cancer cell lysate and 200 μL of 2 mg PLGA nanovaccine loaded with water-insoluble components in 8 M urea were subcutaneously injected on days 42, 35, 28, 21, and 14 before melanoma inoculation. On day 0, 1.5 × 10 5 B16F10 cells.

[0109] The PBS control group was subcutaneously injected with 400 μL PBS on days 42, 35, 28, 21, and 14 before melanoma inoculation. On day 0, 1.5×105 B16F10 cells. Blank nanoparticles + cell lysate control group: 400 μL of blank nanoparticles and an equal amount of free cell lysate loaded with the vaccine were subcutaneously injected on days 42, 35, 28, 21, and 14 before melanoma inoculation. Blank nanoparticles and free cell lysate were injected at different sites. On day 0, 1.5×10 5 B16F10 cells.

[0110] In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0111] (4) Experimental results

[0112] like Figure 8 As shown, the tumors of mice in the vaccine group treated with the modified B16F10 tumor tissue whole cell component loaded during the nano vaccine preparation process disappeared completely after vaccination; while the tumors of mice in the vaccine group treated with the unmodified B16F10 tumor tissue whole cell component loaded disappeared 70% after vaccination; while the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger. In summary, the nano vaccine modified by siliconization and adding charged substances in the present invention has a good preventive effect on melanoma, and because more antigens can be loaded after modification, its preventive effect is better than the nano vaccine group that has not been modified during the preparation process.

[0113] Example 3 Colon cancer tumor tissue and colon cancer whole cell components loaded inside and on the surface of nanoparticles for the treatment of colon cancer

[0114] This example uses mouse colon cancer as a cancer model to illustrate how to prepare a nanovaccine loaded with a mixture of colon cancer tumor tissue and whole cell components of colon cancer cells, and use the vaccine to treat colon cancer.

[0115] In this embodiment, MC38 mouse colon cancer was used as a cancer model. Colon cancer tumor tissue and colon cancer cells were first lysed to prepare water-soluble components and water-insoluble components. Then, organic polymer material PLGA was used as a nanoparticle skeleton material, Bacillus Calmette-Guérin (BCG) was used as an immune adjuvant, and a nano vaccine was prepared by a solvent evaporation method and used to treat colon cancer.

[0116] (1) Lysis of tumor tissue and cancer cells and collection of various components

[0117] Each C57BL / 6 mouse was subcutaneously inoculated with 2 × 10 6 When the tumors grow to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were cut into pieces and ground, and an appropriate amount of pure water was added through a cell filter and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed greater than 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution.

[0118] The cultured MC38 cancer cell line was collected and centrifuged at 350g for 5 minutes, then the supernatant was discarded and washed twice with PBS, then the cells were resuspended in ultrapure water and repeatedly frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at 3000g for 6 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution.

[0119] The water-soluble components from MC38 tumor tissue and MC38 cancer cells and the original water-insoluble components dissolved in 8M urea were mixed in a mass ratio of 1:1 to obtain the raw material source for preparing the vaccine.

[0120] (2) BCG lysis and collection of its components

[0121] The method for lysing BCG and collecting its components are the same as those for cancer cells, except that the MC38 cancer cell line is replaced by BCG.

[0122] (3) Preparation of nano vaccines

[0123] In this embodiment, the nano vaccine and the blank nanoparticles used as the control are prepared by the emulsion method in the solvent evaporation method. The preparation method is the same as that in Example 1, except that the water-soluble component or the water-insoluble component in Example 1 is replaced by the corresponding mixture in this embodiment; the poly(I:C) immune adjuvant is replaced by the BCG lysate of this embodiment. The average particle size of the nano vaccine is about 280nm, and the surface potential of the nano vaccine is about -4.5mV; each 1mg PLGA nanoparticle is loaded with about 290μg protein or polypeptide components, and the total amount of immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.04mg, and half of them are inside and outside. The particle size of the blank nanoparticles is about 260nm. When preparing the blank nanoparticles, pure water or 8M urea containing an equal amount of poly(I:C) is used to replace the corresponding water-soluble components and water-insoluble components.

[0124] (4) Using nano-vaccines to treat colon cancer in mice

[0125] The control groups of this study were PBS group and blank nanoparticles + cell lysate group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice.

[0126] The dosing schedule for the nano-vaccine group was as follows: on day 0, each mouse was subcutaneously inoculated with 2×10 6 MC38 cells were then injected subcutaneously with 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components and 200 μL of 2 mg PLGA nanovaccine loaded with original water-insoluble components dissolved in 8 M urea on days 3, 6, 9, 12, 15, and 18. The PBS control group was as follows: On day 0, 2 × 10 6 MC38 cells were injected subcutaneously on the 3rd, 6th, 9th, 12th, 15th and 18th days. Blank nanoparticles + cell lysate control group: 2×10 MC38 cells were injected subcutaneously on the lower right back of each mouse on day 0. 6 MC38 cells were injected subcutaneously on the 3rd, 6th, 9th, 12th, 15th and 18th days, respectively, with 400 μL of blank nanoparticles and free cell lysate with the same amount of vaccine load. Blank nanoparticles and free cell lysate were injected at different sites. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v=0.52×a×b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0127] (4) Experimental results

[0128] like Fig. 9 As shown, the tumors of mice in the vaccine-treated group loaded with colon cancer tumor tissue and colon cancer cell components that were modified during the preparation of the nanovaccine were partially cured and disappeared after treatment; while the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger. In summary, the nanovaccine modified by siliconization and addition of cationic substances in the present invention has a good therapeutic effect on colon cancer, and because more antigens can be loaded after modification, its therapeutic effect is better than that of the nanovaccine group that was not modified during the preparation process.

[0129] Example 4 Whole cell components of melanoma tumor tissue and lung cancer tumor tissue loaded inside and on the surface of nanoparticles for the treatment of melanoma

[0130] This embodiment uses melanoma as a cancer model to illustrate how to prepare a nano vaccine loaded with whole cell components of melanoma tumor tissue and lung cancer tumor tissue, and uses this vaccine to treat melanoma. In this embodiment, first, B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue are cracked to prepare water-soluble components and water-insoluble components of tumor tissue. Then, organic polymer material PLGA is used as a nanoparticle skeleton material, and manganese particles and CpG are used as immune adjuvants to prepare a nano vaccine loaded with tumor tissue components using a solvent volatilization method. Then, this nano vaccine is used to treat melanoma.

[0131] (1) Lysis of tumor tissue and collection of various components

[0132] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells or 2×10 6 LLC lung cancer cells, when the tumors grew to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. The tumor lysis and component collection methods were the same as in Example 1. The water-soluble components from the B16-F10 tumor tissue and the LLC lung cancer tumor tissue were mixed with the original water-insoluble components dissolved in 8M urea in a mass ratio of 1:1 to obtain the raw material sources for preparing the vaccine.

[0133] (2) Preparation of Nanovaccines

[0134] Nano vaccine and the blank nanoparticles in the present embodiment as a comparison adopt the multiple emulsion method preparation in the solvent evaporation method, and adopt low temperature siliconization technology and two kinds of modification methods of adding charged substances to improve the antigenic load in the nano vaccine preparation process.The nano vaccine of the water-soluble component in the load whole cell component and the nano vaccine of the water-insoluble component in the load whole cell component are prepared respectively when preparing, and then used together when using.The nano particle preparation material PLGA molecular weight adopted is 24KDa-38KDa, and the immunoadjuvant adopted is manganese colloidal particles and CpG and manganese particles are distributed in the nano particle inside and CpG is distributed in the nano vaccine surface.After manganese adjuvant is mixed with the water-soluble component or the water-insoluble component in the whole cell component, adopt the multiple emulsion method to prepare the nano particle of internal load antigen and adjuvant as the first aqueous phase. When preparing the manganese adjuvant, first add 1 mL of 0.3M Na3PO4 solution to 9 mL of normal saline, then add 2 mL of 0.3 M MnCl2 solution, and after standing overnight, Mn2OHPO4 colloidal manganese adjuvant is obtained, and the particle size of the manganese adjuvant is about 13nm. Then the manganese adjuvant is mixed with the water-soluble component (60mg / mL) or the water-insoluble component (60mg / mL) in the whole cell component at a volume ratio of 1:3, and the antigen and manganese adjuvant are loaded into the nanoparticles by the double emulsion method. After internal loading of antigen (lytic component) and adjuvant, 100 mg of nanoparticles were centrifuged at 10000g for 20 min, then resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), then centrifuged at 10000g for 20 min, then resuspended with 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 10 min, then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, resuspended with 3 mL of PBS containing histone (5 mg / mL) and polyarginine (10 mg / mL) for 10 min, then washed by centrifugation at 10000g for 20 min, resuspended with 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000g for 20 min and mixed with 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000g for 20 min and mixed with 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, mL of ultrapure water containing 4% trehalose was resuspended and freeze-dried for 48 h; before vaccine injection, it was resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate component containing CpG adjuvant (protein concentration 50 mg / mL) was added and allowed to react at room temperature for 10 min to obtain a nanovaccine modified by cryosilicification and addition of cationic substances with lysate loaded both inside and outside.The average particle size of the nano vaccine is about 360nm, and the surface potential of the nano vaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 300μg of protein or polypeptide components, the CpG adjuvant used inside and outside each 1mg of PLGA nanoparticles is 0.01mg, and the manganese content loaded inside the particles is 0.005mg.

[0135] The preparation method and steps of the unmodified nanovaccine are basically the same as those of the modified nanovaccine, except that the low-temperature siliconization and cationic substance addition steps are not performed. In the preparation process, the antigen is first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysate component) and manganese adjuvant are loaded inside, the nanoparticles are centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. Before the vaccine is used, it is resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate component containing CpG adjuvant (protein concentration 50 mg / mL) is added and reacted at room temperature for 10 minutes to obtain a nanovaccine loaded with lysate inside and outside. The average particle size of the nanovaccine is about 330nm, and the surface potential of the nanovaccine is about -5mV; each 1mg of PLGA nanoparticles is loaded with about 180μg of protein or polypeptide components, each 1mg of PLGA nanoparticles has 0.01mg of CpG adjuvant, and the manganese content loaded inside the particles is 0.005mg.

[0136] The particle size of the blank nanoparticles is about 300 nm. When preparing the blank nanoparticles, pure water or 8M urea containing equal amounts of manganese adjuvant and CpG adjuvant are used to replace the corresponding water-soluble components and water-insoluble components.

[0137] (3) Nanovaccines for cancer treatment

[0138] The control groups of this study were PBS group and blank nanoparticles + cell lysate group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. The dosing regimen of the nanovaccine group was as follows: on day 0, 1.5×10 5 B16F10 cells were injected subcutaneously on the 3rd, 6th, 9th, 12th, 15th and 18th days, respectively, with 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components and 200 μL of 2 mg PLGA nanovaccine loaded with original water-insoluble components. The PBS control group was as follows: on day 0, 1.5×10 5 B16F10 cells were subcutaneously injected with 400 μL PBS on days 3, 6, 9, 12, 15, and 18. Blank nanoparticles + cell lysate control group: On day 0, 1.5×10 5B16F10 cells were added to the mice, and then 400 μL of blank nanoparticles and an equal amount of free cell lysate loaded with the vaccine were subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days. Blank nanoparticles and free cell lysate were injected at different sites. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v=0.52×a×b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0139] (4) Experimental results

[0140] like Fig.10 As shown, the tumors of mice in the vaccine-treated group loaded with whole cell components of mixed tumor tissues of B16F10 and LLC partially disappeared after vaccination, while the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger. In summary, the nanovaccine loaded with whole cell components of multiple tumor tissues of the present invention has a good therapeutic effect on melanoma, and because more antigens can be loaded after modification, its therapeutic effect is better than that of the nanovaccine group that has not been modified during the preparation process.

[0141] Example 5 Water-soluble components of colon cancer and lung cancer cells loaded inside and on the surface of microparticles for the treatment of colon cancer

[0142] This example uses mouse colon cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with only the water-soluble parts of colon cancer and lung cancer cell components, and use the vaccine to prevent colon cancer.

[0143] In this example, MC38 colon cancer tumor tissue and LLC lung cancer cells were first lysed to prepare water-soluble components and water-insoluble components. Then, organic polymer material PLGA was used as micron particle skeleton material, and manganese particles and poly (I: C) were used as immune adjuvants to prepare micron vaccines loaded with water-soluble components of whole cells.

[0144] (1) Lysis of MC38 colon cancer tumor tissue and LLC lung cancer cells and collection of various components

[0145] The collection and lysis methods of tumor tissue and cancer cells and the collection methods of water-soluble components and water-insoluble components are the same as above. The water-soluble components of the two lysates from colon cancer tumor tissue and lung cancer cells are mixed in a mass ratio of 3:1 to obtain the antigen source for preparing the micron vaccine.

[0146] (2) Preparation of micronized vaccines

[0147] In this embodiment, the preparation of micron vaccine and blank micron particles as a control adopts the emulsion method in the solvent evaporation method. The micron particle preparation material used is the organic polymer material PLGA with a molecular weight of 38KDa-54KDa, and the immune adjuvant used is manganese particles and poly (I: C), and the manganese particles are distributed in the vaccine and poly (I: C) is distributed on the surface of the vaccine. In the preparation process of the micron vaccine emulsion method, two modification methods, low-temperature siliconization technology and adding positively charged substances, are used to increase the antigen loading capacity. In the preparation process, a manganese adjuvant is first prepared, and then the manganese adjuvant is mixed with the water-soluble components in the whole cell component as the first aqueous phase and the emulsion method is used to prepare nanoparticles with internal antigen and adjuvant. When preparing the manganese adjuvant, 1 mL of 0.3 M Na3PO4 solution is first added to 9 mL of normal saline, and then 2 mL of 0.3 M MnCl2 solution is added. After being placed overnight, Mn2OHPO4 colloidal manganese adjuvant is obtained, and the particle size of the manganese adjuvant is about 13nm. Then, the manganese adjuvant was mixed with the water-soluble component (60 mg / mL) in the whole cell fraction at a volume ratio of 1:4, and the antigen and the manganese adjuvant were loaded into the microparticles using the double emulsion method. After internal loading of antigen (lytic component) and adjuvant, 100 mg of microparticles were centrifuged at 10000g for 20 min, then resuspended in 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), then centrifuged at 10000g for 20 min, then resuspended in 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 10 min, then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, resuspended in 3 mL of PBS containing histone (5 mg / mL) and polyarginine (10 mg / mL) for 10 min, then washed by centrifugation at 10000g for 20 min, resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000g for 20 min and mixed with 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000g for 10 min and mixed with 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000g for 20 min and mixed with 10 mL of ultrapure water containing 4% trehalose was resuspended and freeze-dried for 48 hours; before the vaccine was injected, it was resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate containing poly(I:C) adjuvant (protein concentration 50 mg / mL) was added and reacted at room temperature for 10 minutes to obtain a micron vaccine modified by frozen siliconization and addition of cationic substances with lysate loaded inside and outside. The average particle size of the nano vaccine is about 1.5μm, and the surface potential of the nano vaccine is about -4mV; each 1mg of PLGA nanoparticles is loaded with about 320μg of protein or polypeptide components, and the poly(I:C) adjuvant used for each 1mg of PLGA nanoparticles is 0.01mg, and the manganese content loaded inside the particles is 0.005mg.

[0148] The preparation method and steps of the unmodified micron vaccine are basically the same as those of the modified micron vaccine, except that the low-temperature siliconization and cationic substance addition steps are not performed. In the preparation process, the antigen is first loaded inside the micron particles by the double emulsion method. After the antigen (lysate component) and manganese adjuvant are loaded inside, the particles are centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 hours. Before the vaccine is used, it is resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate component containing poly(I:C) adjuvant (protein concentration 50 mg / mL) is added and reacted at room temperature for 10 minutes to obtain a micron vaccine loaded with lysate inside and outside. The average particle size of the micron vaccine is about 1.4 μm, and the surface potential of the micron vaccine is about -5mV; each 1mg of PLGA micron particles is loaded with about 190μg of protein or polypeptide components, each 1mg of PLGA nanoparticles poly(I:C) adjuvant is 0.01mg, and the manganese content loaded inside the particles is 0.005mg.

[0149] The particle size of the blank microparticles is about 300 nm. When preparing the blank microparticles, pure water or 8M urea containing equal amounts of manganese adjuvant and poly(I:C) adjuvant are used to replace the corresponding water-soluble components.

[0150] (3) Micron vaccines for cancer treatment

[0151] The control groups of this study were PBS group and blank microparticles + free lysate group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice. The administration scheme of the micro vaccine group was as follows: on day 0, 2×10 6 MC38 cells were injected subcutaneously on days 3, 6, 9, 12, 15, and 18, respectively, with 200 μL of 4 mg PLGA micro-vaccine loaded with water-soluble components of cancer cell lysate inside and on the surface. The PBS control group was as follows: On day 0, 2×10 6 MC38 cells were injected subcutaneously on days 3, 6, 9, 12, 15, and 18. Blank microparticles + cell lysate control group: 2×10 MC38 cells were injected subcutaneously on the lower right back of each mouse on day 0. 6 MC38 cells were injected subcutaneously on the 3rd, 6th, 9th, 12th, 15th and 18th days, respectively, with 200 μL of blank microparticles and free cell lysate with the same amount of vaccine load. Blank microparticles and free cell lysate were injected at different sites. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v=0.52×a×b2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0152] (4) Experimental results

[0153] like Fig.11 As shown, compared with the PBS blank control group, the blank microparticles + free lysate control group and the micron vaccine administration group, the tumor volume growth rate of mice was significantly slowed down and the survival period of mice was significantly prolonged. Moreover, some mice in the micron vaccine administration group had partial disappearance of tumors after vaccination and recovered. It can be seen that the micron vaccine loaded with water-soluble components of colon cancer tumor tissue and lung cancer cells described in the present invention has a good therapeutic effect on colon cancer, and because more antigens can be loaded after modification, its therapeutic effect is better than that of the micron vaccine group that has not been modified during the preparation process.

[0154] Example 6 8M urea dissolves breast cancer cells and is loaded inside and on the surface of microparticles for the prevention of breast cancer

[0155] This example uses mouse breast cancer as a cancer model to illustrate how to use 8M urea to dissolve whole cell components and prepare a micron vaccine loaded with whole cell components to prevent breast cancer. In this example, 4T1 mouse triple-negative breast cancer is used as a cancer model. First, breast cancer cells are inactivated and denatured, and the whole cell components are dissolved after lysing cancer cells with 8M urea. Then, PLGA is used as the micron particle skeleton material, and CpG and Poly (I: C) are used as immune adjuvants to prepare a micron vaccine loaded with whole cell components using a solvent evaporation method.

[0156] (1) Lysis of cancer cells and collection of components

[0157] The cultured 4T1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The resulting cancer cells were inactivated and denatured by ultraviolet light and high temperature heating, respectively, and then 8M urea aqueous solution was used to lyse the breast cancer cells and dissolve the lysate, which was the raw material source for preparing the vaccine.

[0158] (2) Preparation of micronized vaccines

[0159] In this embodiment, the preparation of micron vaccine and blank micron particles as a control adopts the double emulsion method in the solvent evaporation method, the micron particle preparation material adopted is the organic polymer material PLGA with a molecular weight of 38KDa-54KDa, and the immune adjuvants adopted are CpG and Poly (I: C). In the preparation process of the micron vaccine double emulsion method, three modification methods, namely, low temperature siliconization technology, adding positively charged substances and adding negatively charged substances, are used to increase the antigen loading capacity. During the preparation process, the nanoparticles loaded with antigens and adjuvants were first prepared by the double emulsion method. After the antigens and adjuvants were loaded internally, 100 mg of micronized particles were centrifuged at 10000g for 20 minutes, and then resuspended in 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 10 min, and then fixed at -80°C for 24 h. After centrifugation and washing with ultrapure water, the nanoparticles were resuspended in 3 mL of PBS containing histone (5 mg / mL) and protamine (10 mg / mL) and acted for 10 min, and then centrifuged at 10000g for 20 min to wash, and resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 min, and then centrifuged at 10000g for 20 minutes, and then the nanoparticles were resuspended in 7 mL of PBS and acted for 3 mL of PBS solution containing cell lysate (50 mg / mL) was mixed and acted for 10 min, then centrifuged at 10000g for 20 min, and then resuspended in 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 10 min, then fixed at -80℃ for 24 h, washed by centrifugation with ultrapure water, resuspended in 3 mL of PBS containing polyaspartic acid (5 mg / mL), poly(I:C) (3 mg / mL) and CpG (1 mg / mL) for 10 min, then washed by centrifugation at 10000g for 20 min, resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000g for 20 min and resuspended in 10 mL of ultrapure water containing 4% trehalose and dried for 48 h; washed with 7 mL of PBS before vaccine injection. Resuspend and then add 3 mL of cancer cell lysate components (protein concentration 50 mg / mL) and react at room temperature for 10 minutes to obtain a micron vaccine modified by frozen siliconization, addition of cationic substances and addition of anionic substances, which is loaded with lysate inside and outside. The average particle size of the micron vaccine is about 2.5μm, and the surface potential of the micron vaccine is about -4mV; each 1mg PLGA micron particle is loaded with about 340μg of protein or peptide components, 0.01mg of poly (I: C), and 0.001mg of CpG.

[0160] The particle size of the blank microparticles is about 2.4 μm. When preparing the blank microparticles, 8 M urea containing equal amounts of Poly (I: C) and CpG adjuvants are used to replace the corresponding components.

[0161] (3) Micron vaccines for cancer prevention

[0162] Female BALB / c mice aged 6-8 weeks were selected to prepare 4T1 tumor-bearing mice. The micro-vaccine prevention group was subcutaneously injected with 200 μL of 2 mg PLGA micro-vaccine loaded with whole cell components of tumor tissue on days 35, 28, 21, 14, and 7 before tumor inoculation; on day 0, each mouse was subcutaneously inoculated with 4×10 5 The PBS blank control group was subcutaneously injected with 200 μL PBS on days 35, 28, 21, 14, and 7 before tumor inoculation; on day 0, each mouse was subcutaneously inoculated with 4×10 5 The blank microparticles + cell lysate control group was subcutaneously injected with equal amounts of tumor tissue lysate and 2 mg of PLGA blank microparticles on days 35, 28, 21, 14, and 7 before tumor inoculation; 4×10 5 4T1 cells. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v=0.52×a×b 2 Calculate, where v is the tumor volume, a is the tumor length, and b is the tumor width. In the survival experiment, the tumor volume of mice exceeded 2000mm 3 The mice were considered dead and euthanized.

[0163] (4) Experimental results

[0164] like Fig.12 As shown, compared with the control group, the tumor growth rate of the micron vaccine prevention group was significantly slowed down and the survival period of mice was significantly prolonged. It can be seen that the micron vaccine loaded with whole cell components of cancer cells described in the present invention has a preventive effect on breast cancer.

[0165] Example 7 Tumor tissue and cancer cell whole cell components loaded on mannose-targeted modified nanovaccine for the prevention of cancer metastasis

[0166] This example uses a mouse melanoma mouse lung metastasis cancer model to illustrate how to prepare a nano vaccine loaded with melanoma tumor tissue and cancer cell components, and use the vaccine to prevent cancer metastasis. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.

[0167] In this example, mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 6M guanidine hydrochloride aqueous solution, and then the tumor tissue lysate components and the cancer cell lysate components were loaded into the nanovaccine at a mass ratio of 1:4.

[0168] (1) Lysis of tumor tissue and cancer cells

[0169] After collecting mouse B16F10 melanoma tumor tissues and cultured cancer cells, 6M guanidine hydrochloride was used to lyse and dissolve the whole cell components of the tumor tissue and cancer cells, and then the tumor tissue components and the cancer cell components were mixed at a mass ratio of 1:4.

[0170] (2) Preparation of Nanovaccines

[0171] In this embodiment, the nano vaccine and the blank nanoparticles used as the control are prepared by the double emulsion method in the solvent evaporation method. In the process of preparing the nano vaccine, two modification methods, normal temperature or low temperature siliconization technology and adding positively charged substances, are used to increase the antigen loading capacity. The PLGA molecular weight of the nanoparticle preparation material used is 7KDa-17KDa, and the immune adjuvant used is CpG, and CpG is loaded inside the nanoparticles. The preparation method is as described above. In the preparation process, the antigen and CpG are first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysis component) is loaded inside, 100 mg of nanoparticles are centrifuged at 10000g for 20 minutes, and then the nanoparticles are resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL) and CpG (1 mg / mL), and then centrifuged at 10000g for 20 minutes, and then resuspended with 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 24 h or fixed at room temperature for 10 min and then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, and then resuspended with 3 mL of PBS containing polyaspartic acid (10 mg / mL) and acted for 10 min, and then washed by centrifugation at 12000g for 18 min, and washed with 10 mL of cell lysate (50 mg / mL) The cells were resuspended in PBS solution and allowed to act for 10 min, followed by centrifugation at 10,000 g for 20 min. Then, 10 mL of silicate aqueous solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0) was used and fixed at room temperature for 24 h or fixed at room temperature for 10 min and then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, resuspended in 3 mL of PBS containing protamine (5 mg / mL) and polyethyleneimine (PEI, 10 mg / mL) and acted for 10 min, then washed by centrifugation at 10000 g for 20 min, resuspended in 10 mL of PBS solution of maleimide-PEG2-succinimidyl ester-modified mannose (1 mg / mL) and acted at 4°C for 2 h, then washed by centrifugation at 10000 g for 20 min, then resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 min, then centrifuged at 10000 g for 20 min, and used for 10 mL ultrapure water containing 4% trehalose was resuspended and freeze-dried for 48 hours before use. The average particle size of the nano vaccine is about 320nm, and the surface potential of the nano vaccine is about 2mV; each 1mg PLGA nanoparticle is loaded with about 330μg protein or polypeptide components.

[0172] The particle size of the blank nanoparticles is about 300 nm. When preparing the blank nanoparticles, 6M guanidine hydrochloride containing an equal amount of CpG is used to replace the corresponding cell components.

[0173] (3) Nanovaccines targeting dendritic cells for cancer metastasis

[0174] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare lung cancer tumor-bearing mice. The vaccine group was subcutaneously injected with 200 μL of 4 mg PLGA nanoparticles loaded with lysate on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The PBS blank control group was subcutaneously injected with 400 μL PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The blank nanoparticle + lysate control group was subcutaneously injected with 400 μL blank nanoparticles and an equal amount of free lysate loaded with the vaccine on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. On day 0, each mouse was injected with 3×10 5 The mice were killed on day 15 and their lungs were removed to observe the number of cancer masses formed by melanoma metastasis in the lungs.

[0175] (4) Experimental results

[0176] like Fig.13 As shown, compared with the PBS control group and the blank nanoparticle + lysate control group, the number of cancer metastasis in the nano vaccine group mice was significantly reduced. Moreover, the nano vaccine group treated with low temperature siliconization and addition of cationic substances was better than the nano vaccine group treated with room temperature siliconization and addition of cationic substances. This shows that the active targeted nano vaccine loaded with mixed components of tumor tissue and cancer cells described in the present invention can prevent cancer metastasis.

[0177] Example 8 Pancreatic cancer tumor tissue and colon cancer tumor tissue lysis components loaded inside and on the surface of nanoparticles for the treatment of pancreatic cancer

[0178] This example uses mouse pancreatic cancer as a cancer model to illustrate how to prepare a nanovaccine loaded with pancreatic cancer tumor tissue and colon cancer tumor tissue lysate components, and use the vaccine to treat pancreatic cancer.

[0179] In this embodiment, the lysed components of mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue are loaded on the nano vaccine at a mass ratio of 5:1. First, mouse pancreatic cancer and colon cancer tumor tissues are obtained and lysed to prepare water-soluble components and original water-insoluble components dissolved in 8M urea. When preparing the vaccine, the water-soluble component is a mixture of water-soluble components of pancreatic cancer tumor tissue and water-soluble components of colon cancer tumor tissue at a mass ratio of 5:1; the water-insoluble component is a mixture of water-insoluble components of pancreatic cancer tumor tissue and water-insoluble components of colon cancer tumor tissue at a mass ratio of 5:1; then the water-soluble component and the water-insoluble component mixture are mixed at a mass ratio of 1:1 to obtain a mixture of whole cell components. PLGA is used as the nanoparticle skeleton material, poly (I: C) is used as an immune adjuvant to prepare a nano vaccine and the nano vaccine is used to treat tumors in Pan02 pancreatic cancer tumor-bearing mice.

[0180] (1) Lysis of tumor tissue and collection of various components

[0181] Each C57BL / 6 mouse was inoculated subcutaneously in the axilla with 2 × 10 6 MC38 colon cancer cells or 1×10 6 Pan02 pancreatic cancer cells were inoculated in each mouse and grew to a volume of approximately 1000 mm 3 The method of lysing cancer cells and collecting each component are the same as in Example 1.

[0182] (2) Preparation of Nanovaccines

[0183] The method for preparing the nano vaccine in this embodiment is the same as that in Example 1. The average particle size of the nano vaccine is about 250 nm, and the surface potential of the nano vaccine is about -3 mV; each 1 mg of PLGA nanoparticles is loaded with about 280 μg of protein or polypeptide components, and the poly (I: C) immune adjuvant used inside and outside each 1 mg of PLGA nanoparticles is about 0.02 mg, and half of it is inside and half is outside. The particle size of the blank nanoparticles is about 230 nm, and the blank nanoparticles are prepared by using 8M urea containing an equal amount of poly (I: C) to replace the corresponding cell components.

[0184] (3) Nanovaccines for cancer prevention

[0185] Female C57BL / 6 mice aged 6-8 weeks were selected to prepare pancreatic cancer mice. On day 0, 1×10 6Pan02 cells. The vaccine group was subcutaneously injected with 200 μL of 4 mg PLGA nanoparticles loaded with lysate components on days 3, 6, 6, 12, 15, 18 and 21. The PBS blank control group was subcutaneously injected with 400 μL PBS on days 3, 6, 6, 12, 15, 18 and 21. The blank nanoparticle + lysate control group was subcutaneously injected with 200 μL of 4 mg PLGA blank nanoparticles and free lysate on days 3, 6, 6, 12, 15, 18 and 21. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from day 3. The tumor volume was calculated using the formula v=0.52×a×b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0186] (4) Experimental results

[0187] like Fig.14 As shown, compared with the control group, the tumor growth rate in the nano vaccine group was significantly slowed down and the survival period of mice was significantly prolonged. Moreover, the tumors of some mice disappeared. It can be seen that the nano vaccine loaded with whole cell components of pancreatic cancer and colon cancer tumor tissues described in the present invention has a therapeutic effect on pancreatic cancer.

[0188] Example 9 Whole cell components of liver cancer cells are loaded into nanovaccines with Bacillus Calmette-Guérin (BCG) as adjuvant for the prevention of liver cancer

[0189] This example uses BCG as an immune adjuvant to illustrate how to prepare a nano vaccine loaded with whole cell components of liver cancer cells and use the vaccine to prevent liver cancer. In this example, the liver cancer cells are first lysed and then the water-soluble components and the water-insoluble components are collected respectively. Then, PLGA is used as the nanoparticle skeleton material and BCG is used as the immune adjuvant to prepare the nano vaccine using a solvent evaporation method.

[0190] (1) Lysis of cancer cells and collection of components

[0191] In this example, cancer cell lysis and lysate collection are the same as above.

[0192] (2) BCG lysis and collection of its components

[0193] The method of lysing BCG and collecting and solubilizing the lysate in this example is the same as the lysing method in Example 1, except that the tumor tissue is replaced with BCG.

[0194] (3) Preparation of nano vaccines

[0195] The preparation method and materials used in the nanovaccine in this embodiment are the same as those in Example 1. However, in this embodiment, the immunoadjuvant loaded by the nanovaccine is replaced by BCG instead of poly(I:C). The average particle size of the nanovaccine is about 250nm, and the surface potential of the nanovaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 280μg of protein or polypeptide components, and the total amount of BCG immunoadjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.03mg, and half of them are inside and half are outside. The particle size of the blank nanoparticles is about 230nm, and the blank nanoparticles are prepared by using water containing an equal amount of BCG or 8M urea instead of the corresponding cell components.

[0196] (4) Nano vaccines for the prevention of liver cancer

[0197] Female C57BL / 6 mice were selected as model mice to prepare liver cancer tumor-bearing mice. The vaccine group was subcutaneously injected with 200 μL of 2 mg PLGA nanoparticles loaded with water-soluble components in cancer cell lysates and 200 μL of 2 mg PLGA nanoparticles loaded with original water-insoluble components dissolved in 8 M urea on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The PBS blank control group was subcutaneously injected with 400 μL PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The blank nanoparticle + lysate control group was subcutaneously injected with 400 μL blank nanoparticles and free lysate on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. On day 0, 1×10 6 Hepa1-6 liver cancer cells. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v=0.52×a×b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0198] (4) Experimental results

[0199] like Fig.15 As shown, compared with the control group, the tumor growth rate of the group administered with the nano vaccine was significantly slowed down and the survival period of the mice was significantly prolonged. It can be seen that the nano vaccine of the present invention can prevent liver cancer.

[0200] Example 10 Whole cell components of melanoma tumor tissue loaded inside and on the surface of nanoparticles for the treatment of melanoma

[0201] This embodiment uses mouse melanoma as a cancer model to illustrate how to prepare a nano vaccine loaded with whole cell components of melanoma tumor tissue, and uses the vaccine to treat melanoma. In this embodiment, first, B16F10 melanoma tumor tissue is cracked to prepare water-soluble components and water-insoluble components of tumor tissue. Then, organic polymer material PLGA is used as a nanoparticle skeleton material, poly (I: C) is used as an immune adjuvant, and a solvent volatilization method is used to prepare a nano vaccine loaded with water-soluble components and water-insoluble components of tumor tissue. In this embodiment, the method of siliconization and adding cationic substances is used to increase the load of antigens, and only one round of low-temperature siliconization treatment is performed.

[0202] (1) Lysis of tumor tissue and collection of various components

[0203] The method is the same as in Example 1.

[0204] (2) Preparation of Nanovaccines

[0205] In this embodiment, the nano vaccine and the nanoparticles used as the control are prepared by the double emulsion method in the solvent evaporation method. In the process of preparing the nano vaccine, two modification methods, namely low temperature siliconization technology and adding charged substances, are used to increase the antigen loading capacity. The preparation process is the same as that in Example 1. The average particle size of the nano vaccine is about 250nm, and the surface potential of the nano vaccine is about -3mV; each 1mg of PLGA nanoparticles loads about 280μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, and half of it is inside and half is outside.

[0206] The steps of the method for preparing the nanovaccine with only low-temperature silicification treatment are basically the same as those of the nanovaccine with two modification treatments, except that no charged substance treatment step is added after low-temperature silicification. During the preparation process, the antigen was first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysis component) was loaded inside, the nanoparticles were centrifuged at 10000g for 20 minutes, and then 7mL of PBS was used to resuspend the nanoparticles and mixed with 3mL of PBS solution containing cell lysate (50mg / mL), and then centrifuged at 10000g for 20 minutes. Then, 10mL of silicate aqueous solution (containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0) was used to resuspend, and fixed at room temperature for 10 minutes, and then fixed at -80℃ for 24 hours, and then washed by centrifugation with ultrapure water, and then resuspended with PBS for 10 minutes, and then washed by centrifugation at 10000g for 20 minutes, and resuspended with 10mL of PBS solution containing cell lysate (50mg / mL) for 10 minutes, and then centrifuged at 10000g for 20 minutes and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h; before the vaccine is injected, it is resuspended with 7 mL PBS and then 3 mL of cancer tissue lysate containing immune adjuvant (protein concentration 50 mg / mL) is added and reacted at room temperature for 10 min to obtain a frozen siliconized nanovaccine loaded with lysate inside and outside. The average particle size of the nanovaccine is about 250nm, and the surface potential of the nanovaccine is about -3mV; each 1mg PLGA nanoparticle is loaded with about 210μg protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.02mg, and half is inside and half is outside.

[0207] The steps for preparing the nanovaccine with only cationic addition treatment are basically the same as those for the nanovaccines with two modified treatments, except that there is no low-temperature silicification and only the step of adding charged substances. During the preparation process, the antigen was first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysis component) was loaded inside, the nanoparticles were centrifuged at 10,000 g for 20 minutes, and then the nanoparticles were resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), and then centrifuged at 10,000 g for 20 minutes, and then resuspended with 3 mL of PBS containing protamine (5 mg / mL) and polylysine (10 mg / mL) and acted for 10 min, and then centrifuged at 10,000 g for 20 minutes to wash, and resuspended with 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 min, and then centrifuged at 10,000 g for 20 minutes and resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h; before the vaccine was injected, it was resuspended with 7 mL of PBS and then 3 mL of cancer tissue lysate component containing immune adjuvant (protein concentration 50 mg / mL) was added and acted at room temperature for 10 min to obtain a cationic-modified nanovaccine loaded with lysate inside and outside. The average particle size of the nano vaccine is about 240nm, and the surface potential of the nano vaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 220μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, with half inside and half outside.

[0208] The preparation method of the unmodified nano vaccine is the same as that of Example 1. The average particle size of the nano vaccine is about 230 nm, and the surface potential of the nano vaccine is about -5 mV; each 1 mg of PLGA nanoparticles is loaded with about 180 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles contains about 0.02 mg of poly(I:C) immune adjuvant, half of which is used inside and half outside.

[0209] (3) Nanovaccines for cancer prevention

[0210] The control groups of this study were PBS group, frozen siliconized modified vaccine group and cationic modified vaccine group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice.

[0211] The dosing schedule for the nano-vaccine group was as follows: on day 0, each mouse was subcutaneously inoculated with 1.5 × 10 5B16F10 cells were subcutaneously injected with 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components in the lysate and 200 μL of 2 mg PLGA nanovaccine loaded with original water-insoluble components dissolved in 8 M urea on the 4th, 7th, 10th, 13th, 16th, 19th, 24th and 29th days after melanoma inoculation.

[0212] The PBS control group was administered as follows: On day 0, each mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells were subcutaneously injected with 400 μL PBS on days 4, 7, 10, 13, 16, 19, 24, and 29 after melanoma inoculation.

[0213] In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v =0.52×a×b 2 Calculate, where v is the tumor volume, a is the tumor length, b is the width of the tumor. For animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeded 2000 mm 3 The mice were considered dead and euthanized.

[0214] (4) Experimental results

[0215] like Fig.16 As shown, the tumors of mice in the PBS control group grew quickly and all died within 27 days. The tumor growth rate of mice in each vaccine treatment group was significantly slowed down. About 20% of the mice in the vaccine group that did not undergo any modification during the preparation of the nanovaccine recovered after treatment; about 30% of the mice in the vaccine treatment group that were modified by siliconization or adding cationic modification recovered; and about 50% of the mice in the vaccine treatment group that were modified by siliconization and adding cationic modification recovered. In summary, the nanovaccine modified by siliconization and adding cationic substances in the present invention has a good therapeutic effect on melanoma, and its therapeutic effect is better because it can load more antigens after modification.

[0216] Example 11 Whole cell components of melanoma tumor tissue loaded inside and on the surface of nanoparticles for the treatment of melanoma

[0217] This embodiment uses mouse melanoma as a cancer model to illustrate how to prepare a nano vaccine loaded with whole cell components of melanoma tumor tissue, and uses the vaccine to treat melanoma. In this embodiment, B16F10 melanoma tumor tissue is first cracked to prepare water-soluble components and water-insoluble components of tumor tissue. Then, organic polymer material PLGA is used as a nanoparticle skeleton material, poly (I: C) is used as an immune adjuvant, and a solvent volatilization method is used to prepare a nano vaccine loaded with water-soluble components and water-insoluble components of tumor tissue. In this embodiment, a siliconization or calcification method is used to increase the load of the antigen.

[0218] (1) Lysis of tumor tissue and collection of various components

[0219] The method is the same as in Example 1.

[0220] (2) Preparation of Nanovaccines

[0221] In this embodiment, the nano vaccine and the nanoparticles used as the control are prepared by the double emulsion method in the solvent evaporation method. In the process of preparing the nano vaccine, two modification methods, namely low temperature siliconization technology and adding charged substances, are used to increase the antigen loading capacity. The preparation process is the same as that in Example 1. The average particle size of the nano vaccine is about 250nm, and the surface potential of the nano vaccine is about -3mV; each 1mg of PLGA nanoparticles loads about 280μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, and half of it is inside and half is outside.

[0222] The steps of the preparation method of the calcified nanovaccine are as follows: during the preparation process, the antigen is first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysis component) is loaded inside, the nanoparticles are centrifuged at 10,000 g for 20 minutes, and then 7 mL of PBS is used to resuspend the nanoparticles and mix with 3 mL of PBS solution containing cell lysate (50 mg / mL), and then centrifuged at 10,000 g for 20 minutes, and then resuspended with 10 mL of DMEM culture medium and acted at 4°C for 16 hours, and then 100 μL of CaCl2 (1 mmol) is added and acted at 37°C for 2 hours. The mixture was then centrifuged at 10,000 g for 20 min, washed with ultrapure water, resuspended in 3 mL of PBS containing protamine (5 mg / mL) and polylysine (10 mg / mL) and allowed to react for 10 min, then centrifuged at 10,000 g for 20 min, washed, resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) and allowed to react for 10 min, the supernatant was discarded, the precipitate was resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h; before vaccine injection, it was resuspended in 7 mL of PBS and then 3 mL of cancer tissue lysate component containing immune adjuvant (protein concentration 50 mg / mL) was added and allowed to react at room temperature for 10 min to obtain a nanovaccine loaded with lysate both inside and outside. The average particle size of the nano vaccine is about 250nm, and the surface potential of the nano vaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 230μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, with half inside and half outside.

[0223] The preparation method of the unmodified nano vaccine is the same as that of Example 1. The average particle size of the nano vaccine is about 230 nm, and the surface potential of the nano vaccine is about -5 mV; each 1 mg of PLGA nanoparticles is loaded with about 180 μg of protein or polypeptide components, and each 1 mg of PLGA nanoparticles contains about 0.02 mg of poly(I:C) immune adjuvant, half of which is used inside and half outside.

[0224] (3) Nanovaccines for cancer prevention

[0225] The control groups of this study were PBS group, frozen siliconized modified vaccine group and cationic modified vaccine group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice.

[0226] The dosing schedule for the nano-vaccine group was as follows: on day 0, each mouse was subcutaneously inoculated with 1.5 × 10 5B16F10 cells were subcutaneously injected with 200 μL of 2 mg PLGA nanovaccine loaded with water-soluble components in the lysate and 200 μL of 2 mg PLGA nanovaccine loaded with original water-insoluble components dissolved in 8 M urea on the 4th, 7th, 10th, 13th, 16th, 19th, 24th and 29th days after melanoma inoculation.

[0227] The PBS control group was administered as follows: On day 0, each mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells were subcutaneously injected with 400 μL PBS on days 4, 7, 10, 13, 16, 19, 24, and 29 after melanoma inoculation.

[0228] In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v =0.52×a×b 2 Calculate, where v is the tumor volume, a is the tumor length, b is the width of the tumor. For animal experiment ethics, in the mouse survival test, when the tumor volume of the mouse exceeded 2000 mm 3 The mice were considered dead and euthanized.

[0229] (4) Experimental results

[0230] like Fig.17 As shown, the tumors of mice in the PBS control group grew quickly and all died within 27 days. The tumor growth rate of mice in each vaccine treatment group was significantly slowed down. About 20% of the mice in the vaccine group that did not undergo any modification during the preparation of the nanovaccine recovered after treatment; about 30% of the mice in the vaccine treatment group that were treated with calcification and added cations recovered; and about 50% of the mice in the vaccine treatment group that were treated with both silicification and added cations recovered. In summary, the nanovaccine treated with calcification or silicification in the present invention has a good therapeutic effect on melanoma, and low-temperature silicification and added cation treatment can load more antigens and have better therapeutic effects.

Claims

1. Use of a nano-vaccine system for preventing or treating cancer based on whole cell components of melanoma tumor tissue in the preparation of a vaccine for preventing melanoma, characterized in that: A nano-vaccine system for preventing or treating cancer based on whole cell components of melanoma tumor tissue comprises nanoparticles, whole cell components, immune adjuvants, and modifiers for increasing antigen loading or immunogenicity; the modification is to increase the antigen loading by using silicification and adding cationic substances; the modifiers are derived from the modification process; The whole cell component is a water-soluble component and / or a water-insoluble component of a whole cell; the whole cell component is obtained by lysing the whole cell, or the whole cell component is obtained by processing after lysing the whole cell, or the whole cell component is obtained by processing the whole cell and then lysing it; During the preparation of the nanovaccine, the interior and / or surface of the nanoparticles are modified once or multiple times; the surface of the vaccine system is not connected to a target head with active targeting function or is connected to a target head with active targeting function; the whole cell components are encapsulated in the interior of the nanoparticles and / or loaded on the surface of the nanoparticles; the whole cell components loaded on the surface of the nanoparticles are one or more layers; when the whole cell components loaded on the surface of the nanoparticles are multiple layers, there are modifiers between the layers; the silicification is a silicification treatment using a silicate aqueous solution; the cationic substances are protamine and polylysine.

2. The use according to claim 1, characterized in that: Nanoparticles are particles of nanometer size; the preparation materials of the nanoparticles are organic synthetic polymer materials, natural polymer materials or inorganic materials; the shapes of the nanoparticles are spherical, ellipsoidal, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangle, butterfly-shaped or disc-shaped.

3. The use according to claim 2, characterized in that: The particle sizes of the nanoparticles are independently selected from 1 nm to 1000 nm.

4. The use according to claim 1, characterized in that: The surface of the nanovaccine system can be neutral, negatively or positively charged.

5. The use according to claim 1, characterized in that: In the presence of a modifier, the whole cell components or a mixture thereof and an immune adjuvant are loaded inside and / or on the surface of the nanoparticles to obtain a vaccine system.

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