Whole cell fraction delivery system and uses thereof

Abstract

A delivery system that delivers water-soluble and water-insoluble components of whole cellular components using nanoscale or microscale particles, and its use in the production of vaccines for cancer prevention and treatment. The whole cellular component delivery system consists of nanoscale or microscale particles and whole cellular components carried by the particles, where the whole cellular components are the water-soluble and water-insoluble components of all cells in a cell or tissue. Mutant proteins or peptides produced by cancer in the cellular components are carried by nanoparticles or microparticles. Immunogenic substances produced by these disease-related mutations in whole cellular components can be used in the prevention and treatment of cancer, and can be used to produce vaccines for cancer prevention and / or treatment.

Description

Detailed Description of the Invention

[0001] This application claims priority to a Chinese patent application bearing application number 202010223563 and entitled "Delivery system for whole cell fraction and use thereof," filed with the China Patent Office on March 26, 2020, the entire contents of which are incorporated herein by reference.

[0002] [Technical Field] The present invention is in the field of immunotherapy, and specifically relates to a whole cell fraction delivery system and its use, especially to a whole cell fraction delivery system and its use in the production of vaccines for the prevention and treatment of cancer.

[0003] [Background technology] Immunity is a physiological function of the human body that allows it to distinguish between "self" and "non-self" components, destroy and reject antigenic substances (such as viruses and bacteria) that invade the body, or damaged and tumor cells produced by the body itself, thereby maintaining the body's health. In recent years, immunological technology has developed extremely rapidly, particularly in the field of cancer immunotherapy. As our understanding of cancer continues to improve, it has become clear that the human immune system and various immune cells play an important role in inhibiting the development and progression of cancer.

[0004] In recent years, treatments such as PD-1 antibodies and CAR-T have been approved and introduced into clinical practice, demonstrating favorable clinical efficacy but significant limitations. Currently, cancer immunotherapy based on PD-1 antibodies and CAR-T is only effective in certain patients. For example, CAR-T targets B cell surface-specific antigens such as CD19, CD20, and CD22, which are extremely difficult to find or rarely exist in solid tumors. Therefore, treatments such as CAR-T can currently only be used to treat hematologic tumors.

[0005] To improve the treatment of solid tumors and other diseases, scientists in the United States and Germany have adopted new techniques to analyze and identify cancer-specific or cancer-associated antigen polypeptides from tumor cells of cancer patients, then artificially synthesize them in vitro to produce cancer treatment vaccines for use in cancer treatment. This technique has shown some effectiveness in clinical trials with cancer patients. However, this method is time-consuming, labor-intensive, and expensive. Furthermore, the only method currently used is to extract and analyze the water-soluble components of cancer cells to identify polypeptides that differentiate them from normal cells. This method is therefore limited in its use, as it can only identify a limited number of water-soluble antigen polypeptides. Furthermore, many antigenic proteins and polypeptides that are highly immunogenic in the human body are insoluble in pure water and exist in the body by binding to proteins, adsorbing, or localizing on membranes or membrane surfaces. Therefore, these water-insoluble proteins and polypeptides are extremely important. However, there is currently no method for using whole cell fractions of cancer cells as vaccines for cancer prevention and treatment.

[0006] Summary of the Invention In view of the above, an object of the present invention is to provide a delivery system for a whole cell fraction containing a water-soluble component and a water-insoluble component that is insoluble in pure water or an aqueous solution that does not contain a solubilizing agent, and use thereof, in view of the problems existing in the prior art.

[0007] In order to achieve the objectives of the present invention, the present invention adopts the following technical solutions:

[0008] A whole cell fraction delivery system comprising nanoscale or microscale particles and whole cell fractions carried on the particles, the whole cell fractions being the water-soluble and water-insoluble components of all cells in a cell or tissue.

[0009] In the delivery system according to the present invention, the loading method is that the water-soluble and water-insoluble components of whole cells are loaded separately or together inside the particles and / or separately or together on the particle surface, including, but not limited to, loading the water-soluble components together in the particles and on the particle surface, loading the water-soluble components together in the particles and on the particle surface, loading the water-soluble components in the particles and on the water-insoluble components, loading the water-soluble components in the particles and on the particle surface, loading the water-soluble and water-insoluble components in the particles and on the particle surface only the water-soluble components, loading the water-soluble and water-insoluble components in the particles and on the particle surface only the water-soluble components, loading the water-soluble components in the particles and on the particle surface only the water-soluble components, loading the water-soluble components in the particles and on the particle surface both the water-soluble and water-in ... and water-insoluble components in the particles and on the particle surface both the water-soluble and water-insoluble components, loading the water-soluble and water-insoluble components in the particles and on the particle surface both the water-soluble and water-insoluble components, loading the water-soluble and water-insoluble components in the particles and on the particle surface both the water-soluble and water-insoluble components, loading the water-soluble and water-insoluble components in the particles and on the particle surface both the water-soluble and water-insoluble components, loading the water-soluble and water-insoluble components in the particles and on the particle surface both the water-soluble and water-insoluble components.

[0010] In some embodiments, the particles in the delivery system further comprise an immune-enhancing adjuvant inside and / or on the surface thereof.

[0011] The methods of adding the immune enhancing agent include being carried within nanoparticles or microparticles, being carried on the surface of nanoparticles or microparticles, or being carried both within nanoparticles or microparticles and on the surface of nanoparticles or microparticles.

[0012] In the delivery system of the present invention, the whole cell fraction can be divided into two parts, a water-soluble component and a water-insoluble component, based on their solubility in pure water or an aqueous solution containing no solubilizer. The water-soluble component is the original water-soluble portion that is soluble in pure water or an aqueous solution containing no solubilizer, while the water-insoluble component is the original water-insoluble portion that is insoluble in pure water and is converted into a portion that is soluble in an aqueous solution containing a solubilizer or an organic solvent using an appropriate solubilization method. The water-soluble and water-insoluble portions of the whole cell fraction can be solubilized using a solubilizing aqueous solution containing a solubilizer or an organic solvent.

[0013] In the delivery system according to the present invention, the solubilizing agent is at least one solubilizing agent capable of increasing the solubility of a protein or polypeptide in an aqueous solution, and the organic solvent is an organic solvent capable of dissolving the protein or polypeptide.

[0014] In the delivery system according to the present invention, the solubilizer may be urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, alkaline solutions with a pH of 7 or higher, acidic solutions with a pH of less than 7, various proteolytic enzymes, albumin, lecithin, highly concentrated inorganic salts, Triton, Twain, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acids, glucosides, choline, Brij TM Examples of suitable water-insoluble components include, but are not limited to, octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630, etc. Those skilled in the art will appreciate that other methods capable of solubilizing proteins and polypeptide fragments can also be employed to convert the water-insoluble components from insoluble to soluble in pure water.

[0015] In the delivery system according to the present invention, the organic solvent includes, but is not limited to, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, and ethyl acetate. Those skilled in the art will appreciate that other organic solvents can also be used, including those capable of solubilizing proteins and polypeptide fragments.

[0016] In the delivery system described in the present invention, the immune-enhancing adjuvant includes at least one of, but is not limited to, immune-enhancing agents derived from microorganisms, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, herbal medicines, and others.

[0017] In the present invention, the immune-enhancing adjuvant may include a pattern recognition receptor agonist, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extract residue, BCG muramyl dipeptide, Mycobacterium phlei, polyactin A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virus particles, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, new bovine liver active peptide, miquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly ICLC, Corynebacterium parvum These include, but are not limited to, at least one of the active ingredients of the following: flu vaccine, hemolytic streptococcal 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, and astragalus.

[0018] Those skilled in the art will appreciate that the immune-enhancing adjuvant can also employ other substances capable of enhancing the immune response.

[0019] In the delivery system described herein, the nanoscale-sized particles have a diameter of 1 nm to 1000 nm. In some embodiments, the nanoscale-sized particles have a diameter of 50 nm to 800 nm. Furthermore, in some embodiments, the nanoscale-sized particles have a diameter of 100 nm to 600 nm.

[0020] In the delivery system described herein, the micro-sized particles have a diameter of 1 μm to 1000 μm. In some embodiments, the micro-sized particles have a diameter of 1 μm to 100 μm. In some embodiments, the micro-sized particles have a diameter of 1 μm to 10 μm. Furthermore, in some embodiments, the micro-sized particles have a diameter of 1 μm to 5 μm.

[0021] In the delivery system according to the present invention, the surface of said nano-sized particles or micro-sized particles may be electrically neutral, negatively charged or positively charged.

[0022] In the delivery system according to the present invention, the nano- or micro-sized particles are made from organic synthetic polymer materials, natural polymer materials or inorganic materials.

[0023] Here, the organic synthetic polymer material is a biocompatible or degradable polymer material, including, but not limited to, PLGA, PLA, PGA, poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acids, and synthetic polypeptides.

[0024] The natural polymeric material is a biocompatible or degradable polymeric material, and includes, but is not limited to, lecithin, cholesterol, starch, sugars, polypeptides, sodium alginate, albumin, collagen, gelatin, cell membrane components, and the like.

[0025] The inorganic materials are non-specifically biotoxic materials, including, but not limited to, ferric oxide, ferric oxide, calcium carbonate, and calcium phosphate.

[0026] The shape of the delivery system according to the present invention may be any general shape, including, but not limited to, a sphere, an oval, a barrel, a polygon, a rod, a sheet, a wire, a worm, a square, a triangle, a butterfly, or a disk.

[0027] The delivery system according to the present invention can be prepared according to the developed manufacturing methods of nano-sized particles and micro-sized particles, including, but not limited to, the common solvent evaporation method, dialysis method, extrusion method, and hot melt method. In some embodiments, the delivery system is prepared using the double emulsion method in the solvent evaporation method.

[0028] The whole cell fraction delivery system described in the present invention can deliver the loaded whole cell fraction to relevant immune cells, and activate and enhance the killing effect of the autoimmune system against cancer cells due to the immunogenicity of the loaded components. Therefore, the present invention further provides the use of the whole cell fraction delivery system in the manufacture of a vaccine for the prevention and / or treatment of cancer.

[0029] When the whole cell fraction delivery system described in the present invention is used to prevent or treat a disease, it is possible to use nanoparticles or microparticles carrying only water-soluble components and nanoparticles or microparticles carrying only water-insoluble components simultaneously, to use nanoparticles or microparticles carrying only water-soluble components, to use nanoparticles or microparticles carrying only water-insoluble components, or to use nanoparticles or microparticles carrying both water-soluble and water-insoluble components.

[0030] As can be seen from the above technical solutions, the present invention provides a delivery system for delivering water-soluble and water-insoluble components of cells using nanoscale or microscale particles, and its use in the production of vaccines for cancer prevention and treatment. The whole cellular fraction of relevant cells or tissues is divided into two parts based on their solubility in pure water: a water-soluble component that dissolves in pure water and a water-insoluble component that does not dissolve in pure water. The water-soluble and water-insoluble components are then loaded onto nanoparticles or microparticles, thereby allowing most of the mutated proteins and polypeptides caused by cancer in the cellular fraction to be loaded onto the nanoparticles or microparticles. The water-soluble and water-insoluble components of the cellular fraction encompass the entire cellular fraction, and the water-soluble and water-insoluble parts of the cellular fraction can also be simultaneously solubilized using an aqueous solution containing a solubilizing agent. Unmutated proteins, polypeptides, and genes that are the same components as those in normal cells do not provoke an immune response due to immune tolerance generated during the development of the autoimmune system. However, mutations in genes, proteins, and polypeptides caused by cancer, etc., are immunogenic and activate an immune response due to the lack of immune tolerance generated during the development of the autoimmune system. These immunogenic substances produced by disease mutations in whole cell fractions can be used to treat cancer.

[0031] The whole cell fraction delivery system described in the present invention can be used to produce vaccines for the prevention and / or treatment of cancer. The cancers can be of all types, including hematological and solid tumors, including endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, respiratory system tumors, blood cancer, skin cancer, breast cancer, lung cancer, liver cancer, stomach cancer, pancreatic cancer, brain tumors, colon cancer, prostate cancer, rectal cancer, head and neck cancer, kidney cancer, bone cancer, nasal cancer, bladder cancer, thyroid cancer, esophageal cancer, cervical cancer, ovarian cancer, uterine cancer, pelvic cancer, testicular cancer, penile cancer, lymphoma, tongue cancer, gum cancer, retinoblastoma, and sarcoma. When used as a cancer vaccine for the prevention and treatment of cancer, the vaccine described in the present invention can be administered several times before or after the onset of cancer to activate the body's immune system, delay cancer progression, treat cancer, prevent cancer onset, prevent cancer metastasis, or prevent cancer recurrence.

[0032] [Drawing Description] In order to more clearly describe the embodiments of the present invention or the technical solutions in the prior art, the drawings that need to be used to describe the embodiments or the prior art will be briefly described below.

[0033] Figure 1 is a schematic diagram of the manufacturing process and field of use of the vaccine described in the present invention, where a: is a schematic diagram of collecting and manufacturing nanovaccines or microvaccines from water-soluble and water-insoluble components, respectively, and b: is a schematic diagram of dissolving the whole cell fraction using a solubilizing solution containing a solubilizing agent and manufacturing nanovaccines or microvaccines.

[0034] Figures 2 to 17 are schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle.

[0035] Figures 18 to 21 show the experimental results of the effects of nanovaccines in Examples 1 to 3, respectively, on the tumor growth rate and survival time of mice when used to treat melanoma; a, experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b, experiment on the effect of nanovaccine treatment on the survival time of tumor-inoculated mice (n≧8), where each data point is the mean ± standard error (mean ± SEM); the significant differences in the tumor growth inhibition experiment in Figure a were analyzed using ANOVA, and the significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference between the group and the PBS blank control group at p<0.05; ☆ indicates a significant difference between the group and the blank nanoparticles + cell lysate + PD-1 antibody control group at p<0.05.

[0036] 22 shows the experimental results of the effects of nanovaccines in Examples 4 and 5 on tumor growth rate and survival time in mice when used to prevent melanoma; a, Experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b, Experiment on the effect of nanovaccine treatment on survival time of tumor-inoculated mice (n≧8). Each data point is the mean±SEM. The significance of the tumor growth inhibition experiment in Figure a was analyzed using ANOVA, and the significance of the significance of the experiment in Figure b was analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference of p<0.05 compared to the PBS blank control group.

[0037] Figures 23 to 28 show experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11, respectively, on tumor growth rate and survival time in mice when used for the prevention or treatment of breast cancer. a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7). b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Each data point represents the mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group. ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group.

[0038] [Specific implementation method] The present invention discloses a delivery system for whole cell fractions and its use. Those skilled in the art can realize this by appropriately improving the process parameters with reference to the contents of this specification. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and product of the present invention have been described through preferred embodiments, but it is clear that those skilled in the art can make modifications or appropriate modifications and combinations to the methods described herein to realize and use the present invention without departing from the content, spirit, and scope of the present invention.

[0039] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0040] The whole cell fraction delivery system comprises nanoscale or microscale particles and whole cell fractions loaded onto the particles, the whole cell fractions being the water-soluble and water-insoluble components of all cells in a cell or tissue.

[0041] In the present invention, after lysing cancer cells or tissues, first obtain water-soluble components that are soluble in pure water or an aqueous solution that does not contain a solubilizing agent, then dissolve the water-insoluble components in the solubilizing solution using an aqueous solubilizing solution containing a solubilizing agent, thereby converting all cellular fractions into fractions that can be dissolved in aqueous solution, which are then carried on the inside or outside of nanoparticles or microparticles to produce nanovaccines or microvaccines for use in cancer prevention and treatment.In actual use, after lysing the cells or tissues, the entire cellular fraction is directly dissolved in an aqueous solubilizing solution containing a solubilizing agent without separately recovering the water-soluble and water-insoluble components, and the entire cellular fraction dissolved in the solubilizing solution is used to produce nanovaccines or microvaccines.

[0042] The present invention uses an aqueous solution containing a solubilizer to solubilize a fraction insoluble in pure water or an aqueous solution not containing a solubilizer into a specific solubilizer solution, which is then used to produce nanoparticles and microparticles, thereby improving the inclusion and immunogenicity of antigenic substances or components carried by the nanoparticles or microparticles.

[0043] The present invention separates the whole cell fraction of cancer cells or tumor tissue into a water-soluble portion that can be dissolved in pure water or an aqueous solution that does not contain a solubilizing agent, and a water-insoluble portion that can be dissolved in an aqueous solution of a specific solubilizing agent, and by supporting the water-soluble portion and the water-insoluble portion in nanoparticles or microparticles and on their surfaces, it is ensured that the produced vaccine contains most of the antigenic substance.

[0044] The water-soluble and water-insoluble portions of the cellular fractions encompass whole-cell components and fractions. Non-mutated proteins, peptides, and genes identical to normal cellular components do not provoke an immune response due to the immune tolerance generated during the development of the autoimmune system, whereas mutations in genes, proteins, and peptides generated by cancer lack the immune tolerance generated during the development of the autoimmune system, making them immunogenic and capable of activating the body's immune response against cancer cells. The use of cancer cell-specific immunogenic substances produced by these disease-related mutations in the whole-cell fractions can be used for the prevention and treatment of cancer.

[0045] To enhance the immunogenicity and efficacy of the vaccine, immune enhancing agents with certain immunomodulatory functions can also be added.

[0046] The whole cell fraction delivery system described in the present invention can be used to produce vaccines for cancer prevention and / or treatment, and its production process and application field are as shown in Figure 1. In the production, after lysing cells or tissues, the water-soluble and water-insoluble components can be first collected to produce nanovaccines or microvaccines, respectively; alternatively, a solubilizing solution containing a solubilizing agent can be directly used to lyse cells or tissues, and then the whole cell fraction can be dissolved to produce nanovaccines or microvaccines.

[0047] The whole cell fractions described in the present invention can be used to prepare nanovaccines or microvaccines after inactivation or denaturation treatment before or (and) after lysis, or they can be used directly to prepare nanovaccines or microvaccines without inactivation or denaturation treatment before or (and) after cell lysis. In some embodiments of the present invention, tumor tissue cells are inactivated or (and) denatured before lysis. However, in actual use, inactivation or (and) denaturation treatment can be performed after cell lysis, or both before and after cell lysis. In some embodiments of the present invention, the inactivation or (and) denaturation treatment method before or (and) after cell lysis is ultraviolet irradiation and high-temperature heating. In actual use, inactivation or denaturation treatment methods such as irradiation with radioactive materials, high pressure, freeze-drying, and formaldehyde can also be used. Those skilled in the art will understand that in actual use, they may make appropriate adjustments according to specific conditions.

[0048] The structural schematic diagrams of the whole cell fraction delivery system according to the present invention are shown in Figures 2 to 17. In the actual use process, only one of the nanoparticles or microparticles with a specific structure may be used, or two or more nanoparticles or microparticles with different structures may be used simultaneously.

[0049] In an embodiment, the immune enhancing agent is carried both within the nanoparticles or microparticles and on the surface of the nanoparticles or microparticles, and in the actual use process, the immune enhancing agent may be carried only within the nanoparticles or microparticles, or only on the surface of the nanoparticles or microparticles, or no immune enhancing agent may be added.

[0050] In some embodiments, as shown in Figure 2, the present invention first solubilizes the water-soluble and / or water-insoluble portions of the cell fraction, which are soluble in pure water, with a solubilizing agent, and then loads them into nanoparticles or microparticles, along with the immunopotentiator; then adsorbs the water-soluble and / or water-insoluble portions of the cell fraction onto the surface of the nanoparticles, along with the immunopotentiator. In this way, the loading capacity of the water-soluble or water-insoluble components of cells in the nanoparticles or microparticles can be maximized. In actual use, a solubilizing solution containing a solubilizing agent (e.g., an 8M urea aqueous solution or a 6M guanidine hydrochloride aqueous solution) can also be used directly to directly dissolve cells or tissues, and then the whole cell fraction can be directly dissolved before producing a nanovaccine or microvaccine.

[0051] The method for producing nanovaccines and microvaccines according to the present invention is a general production method. The manufacturing method can be a solvent evaporation method. In some embodiments, the nanovaccine or microvaccine The production of nanoparticles was carried out by the double emulsion method in the solvent evaporation method. or microparticles The manufacturing material is an organic polymer, polylactic-co-glycolic acid (PLGA), with a molecular weight of 24KDa-38KDa. The immunoadjuvants used are poly(I:C), Bacillus Calmette-Guerin (BCG), or CpG. Those skilled in the art will be able to determine the manufacturing method, manufacturing process, and nanoparticles used according to their specific conditions in the actual application process. or microparticles It is understood that the manufacturing materials, the type and concentration of the immunoadjuvant, etc. can be appropriately adjusted.

[0052] In some embodiments, the specific preparation method of the double emulsion method employed in the present invention is as follows.

[0053] Step 1: Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a medical polymer material at a second predetermined concentration.

[0054] In some embodiments, the aqueous phase solution contains each fraction of a cancer cell lysate and the immunopotentiating adjuvant poly(I:C), BCG, or CpG, where each fraction of the cancer cell lysate is the original water-soluble component or the original water-insoluble component dissolved in 8 M urea during production. The concentration of the cancer cell-derived water-soluble component contained in the aqueous phase solution or the original water-insoluble component dissolved in 8 M urea during production, i.e., the first predetermined concentration, requires a protein polypeptide concentration content of greater than 1 ng / mL. The reason for selecting such a concentration is that, generally, the higher the concentration of the cell lysate aqueous solution used, the greater the amount of various cancer antigens loaded onto the nanoparticles produced. The inventors have found through extensive experiments that when the concentration of the produced protein polypeptides exceeds 1 ng / mL, i.e., the first predetermined concentration exceeds 1 ng / mL, sufficient cancer antigens can be loaded and the relevant immune response can be activated. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng / mL. The first predetermined volume is 600 μL.

[0055] In some embodiments, the aqueous phase solution contains tumor tissue lysate fractions and the immunopotentiating adjuvant poly(I:C), BCG, or CpG, where each fraction in the tumor tissue lysate is the original water-soluble component or the original water-insoluble component dissolved in 8 M urea during production. The concentration of the tumor tissue-derived water-soluble component contained in the aqueous phase solution or the original water-insoluble component dissolved in 8 M urea during production, i.e., the first predetermined concentration, requires a protein polypeptide concentration content of greater than 1 ng / mL. The reason for selecting such a concentration is that the higher the concentration of the tumor tissue lysate aqueous solution used, the greater the amount of various cancer antigens loaded onto the nanoparticles produced. The inventors have found through extensive experiments that when the concentration of the produced protein polypeptides exceeds 1 ng / mL, i.e., the first predetermined concentration exceeds 1 ng / mL, sufficient cancer antigens can be loaded and the relevant immune response can be activated. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng / mL. The first predetermined volume is 600 μL.

[0056] In the present invention, a medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing the medical polymer material at a second predetermined concentration. In some embodiments, the medical polymer material is PLGA, the organic solvent is dichloromethane, and the volume of the resulting organic phase, i.e., the second predetermined volume, is 2 mL. Furthermore, 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.

[0057] In this invention, PLGA is selected because it is a biodegradable material and is FDA approved for use as a drug dressing. Studies have shown that PLGA has certain immunomodulatory functions, making it suitable as an adjuvant for vaccine production.

[0058] In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase, and in the present invention, the ratio range of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1:1.1 to 1:5000, preferably 1:10. In a specific implementation process, the size of the produced nanoparticles can be adjusted by adjusting the first predetermined volume, the second predetermined volume, and the ratio between the first predetermined volume and the second predetermined volume as needed.

[0059] Step 2: The mixed solution obtained in Step 1 is subjected to ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute.

[0060] The purpose of this step is to carry out nanometerization, and the length of ultrasonic time or the stirring speed and time can control the size of the produced nanoparticles. If it is too long or too short, the particle size will change, so it is necessary to select an appropriate ultrasonic time. In the present invention, the ultrasonic time is more than 2 seconds, the stirring speed is more than 50 rpm, and the stirring time is more than 1 minute.

[0061] Step 3: The mixture obtained after the treatment in Step 2 is added to a third predetermined volume of an aqueous solution containing an emulsifier at a third predetermined concentration, and ultrasonicated for more than 2 seconds or stirred for more than 1 minute.

[0062] In this step, the mixture obtained in step 2 is added to an aqueous solution of an emulsifier, followed by ultrasonication or stirring to nanometerize it.

[0063] 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 ratio between the second predetermined volume and the third predetermined volume is set to a range of 1:1.1 to 1:1000, preferably 2:5. In a specific implementation process, the ratio between the second predetermined volume and the third predetermined volume can be adjusted to control the size of the nanoparticles.

[0064] Similarly, the ultrasonic wave time or stirring time in this step, and the volume and concentration of the emulsifier aqueous solution are all determined to obtain nanoparticles of an appropriate size. or microparticles Take the value to get

[0065] Step 4: The liquid after the treatment in Step 3 is added to a fourth predetermined volume of an emulsifier aqueous solution having a fourth predetermined concentration, and stirred until predetermined stirring conditions are met.

[0066] In this step, the aqueous emulsifier solution is still PVA, and the fourth predetermined volume is greater than 50 mL.

[0067] The fourth predetermined concentration is 5 mg / mL, and the fourth predetermined concentration is selected to obtain nanoparticles of an appropriate size. The fourth predetermined volume is selected according to the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the fourth predetermined volume to the third predetermined volume ranges from 1:1.5 to 1:2000, preferably 1:10. In a specific implementation process, nanoparticles or microparticles The ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the size of the third predetermined volume.

[0068] In the present invention, the predetermined stirring conditions in this step are until the evaporation of the organic solvent is complete, that is, until the evaporation of dichloromethane in step 1 is complete.

[0069] Step 5: The mixture that satisfies the predetermined stirring conditions of Step 4 is centrifuged at a speed exceeding 100 RPM for at least 1 minute, the supernatant is removed, and the remaining precipitate is resuspended in a fifth predetermined volume of an aqueous solution containing a fifth predetermined concentration of a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).

[0070] In some embodiments of the present invention, the precipitate obtained in step 5 does not need to be lyophilized if it is resuspended in a sixth predetermined volume of PBS (or saline), and the subsequent nanoparticles or microparticles Experiments related to the adsorption of cancer cell lysates onto the surface can be directly performed.

[0071] In some embodiments of the present invention, the precipitate obtained in step 5 needs to be lyophilized when resuspended in an aqueous solution containing a lyoprotectant, and after lyophilization, the nanoparticles or microparticles We will conduct experiments related to the adsorption of cancer cell lysate onto the surface of

[0072] In the present invention, the lyoprotectant is selected to be trehalose.

[0073] In the present invention, the fifth predetermined volume of the lyoprotectant in this step is 20 mL, and the fifth predetermined concentration is 4% by mass, in order not to affect the lyoprotectant effect during subsequent lyoprotection.

[0074] Step 6: The suspension containing the lyoprotectant obtained in step 5 is freeze-dried, and the freeze-dried product is stored for use.

[0075] Step 7: A sixth predetermined volume of the PBS (or saline) obtained in step 5 containing the nanoparticles suspended therein is added. or containing microparticles Suspension or freeze-dried nanoparticles obtained in step 6 using a sixth predetermined volume of PBS (or saline) or microparticles The lyophilized material containing the lyophilized material and the lyophilization protectant is suspended and mixed with a predetermined volume of the water-soluble component or the original water-insoluble component dissolved in 8M urea, and then allowed to stand for at least 1 minute to obtain a nanovaccine or microvaccine.

[0076] In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, preferably 1:100 to 100:1, and most preferably 1:30 to 30:1.

[0077] In some embodiments, the suspended nanoparticles or microparticlesThe volume of the suspension is 10 mL, and the volume of the aqueous component containing the cancer cell lysate or the tumor tissue lysate or the original insoluble component dissolved in 8 M urea is 1 mL.

[0078] In the present invention, the water-soluble components including cancer cell lysate or tumor tissue lysate used, or the original water-insoluble components dissolved in 8M urea, contain poly(I:C), Bacillus Calmette-Guérin vaccine (BCG), or CpG, and the concentration of poly(I:C), BCG, or CpG exceeds 1 ng / mL.

[0079] The particle size of nanovaccines or microvaccines is nanoscale or microscale, and the particle size is within an appropriate range to ensure that the vaccine is phagocytosed by antigen-presenting cells and improve phagocytosis efficiency. The particle size of nanovaccines is 1 nm to 1000 nm, more preferably 30 nm to 1000 nm, and most preferably 100 nm to 600 nm; the particle size of microvaccines is 1 μm to 1000 μm, more preferably 1 μm to 100 μm, more preferably 1 μm to 10 μm, and most preferably 1 μm to 5 μm. In this embodiment, the particle size of nanovaccines is 100 nm to 600 nm, and the particle size of microvaccines is 1 μm to 5 μm.

[0080] In addition, although urea and guanidine hydrochloride are used in the present invention to solubilize the original water-insoluble components in cancer cell lysate or tumor tissue lysate, other solubilizing agents that solubilize the original water-insoluble components in cancer cell lysate or tumor tissue lysate in an aqueous solution can be used in practice, such as sodium deoxycholate, SDS, alkaline solution with a pH greater than 7, acidic solution with a pH less than 7, albumin, lecithin, highly concentrated inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM-35, octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, IGEPAL® CA-630, or the above solubilizers can be used to simultaneously dissolve water-soluble and water-insoluble components.

[0081] 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, and in actual use, other urea concentrations or guanidine hydrochloride concentrations that solubilize the original water-insoluble components in the cancer cell lysate or tumor tissue lysate in aqueous solution can be used; or 8M urea aqueous solution can be used to simultaneously dissolve the water-soluble and water-insoluble components.

[0082] Furthermore, in the present invention, a nanovaccine or microvaccine The preparation of nanoparticles or microparticles employs a double emulsion method, and in fact, other commonly used nanoparticle or microparticle preparation methods can be employed.

[0083] Furthermore, in the present invention, a nanovaccine or microvaccine The manufacturing material is PLGA, but in fact any other material capable of manufacturing nanoparticles or microparticles can be employed.

[0084] 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 8M urea are respectively carried inside the nanoparticles and adsorbed onto the surface of the nanoparticles. In actual use, the water-soluble components in the cancer cell lysate or tumor tissue lysate and the original water-insoluble components dissolved in 8M urea are mixed to form nanoparticles. or microparticles Internally supported or nanoparticles or microparticlesor 8M urea can be used to simultaneously dissolve the water-soluble and water-insoluble components and allow them to be loaded inside and / or adsorbed onto the surface of the nanoparticles or microparticles.

[0085] In addition, although poly(I:C), Bacillus Calmette-Guerin vaccine (BCG), and CpG are used as immunoadjuvants in the present invention, in practice, no immunoadjuvant may be added, or other immunoadjuvants with immunopotentiating functions may be added, such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanol extract residue, BCG muramyl dipeptide, Mycobacterium phlei, polyactin A, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus, cholera enterotoxin, saponin and derivatives thereof, resiquimod (Resiquimod). Active ingredients in traditional Chinese medicines include quimod, thymosin, neonatal bovine liver active peptide, miquimod, polysaccharides, curcumin, immune adjuvant polyICLC, Corynebacterium parvum, hemolytic streptococcus preparations, 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, and astragalus.

[0086] Furthermore, in the present invention, the vaccine used in some embodiments is a nanovaccine, and in some embodiments is a microvaccine, and those skilled in the art can choose to use a nanovaccine or a microvaccine according to the actual situation.

[0087] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described in combination with the examples of the present invention, but obviously, the described examples are only a part of the examples of the present invention, and are not all of the examples. Based on the examples of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0088] Unless otherwise specified, all methods used in the examples of the present invention are conventional methods; materials, reagents, etc. used can be commercially available. The structures, manufacturing methods, disease treatment strategies, combination strategies with other immunotherapeutic agents, combination strategies with other targeted therapeutic agents, etc. of nano- or micro-sized particles included in the embodiments of the present invention are merely representative methods. Other nano- or micro-sized particle structures, manufacturing methods, disease treatment strategies, combination strategies with other immunotherapeutic agents, and combination strategies with other targeted therapeutic agents can also be used with the methods described in the present invention. Although the examples only list the use of the present invention in some cancers, the present invention can also be used in other cancers. Regarding the specific methods or materials used in the examples, those skilled in the art can make conventional alternative choices based on the technical concept of the present invention and existing technology, and are not limited to the specific descriptions in the examples of the present invention.

[0089] PD-1 antibodies have been approved for the treatment of many cancers due to their excellent clinical efficacy. Therefore, in the embodiments of the present invention, in addition to using the nano- or micro-vaccine to treat cancer, the applicant also tested the use of the nano- or micro-vaccine in combination with a PD-1 antibody to treat cancer. In actual use, the specific administration time, administration frequency, administration method, and combination with other drugs can be adjusted according to the circumstances.

[0090] Example 1: Whole cell fractions of melanoma cancer cells are loaded into and onto nanoparticles for cancer treatment. In this example, a method for treating melanoma using a mouse melanoma as a cancer model, producing a nanovaccine carrying a whole cell fraction, and using the vaccine was described.

[0091] PD-1 antibodies have been approved for the treatment of many cancers due to their excellent clinical efficacy. Therefore, in this example, in addition to using the nanovaccine only to treat melanoma, the applicant also tested the combination of the nanovaccine and PD-1 antibodies to treat melanoma. In actual use, the specific administration time, frequency, and administration method can be adjusted according to the situation.

[0092] In this example, B16F10 mouse melanoma cells were used as a cancer cell model. First, B16F10 cells were dissolved to produce water-soluble and water-insoluble components of B16F10 cells. Next, a nanovaccine was prepared by solvent evaporation using the organic polymer PLGA as the nanoparticle scaffold and polyinosinic-polycytidylic acid (Poly(I:C)) as the immunoadjuvant, carrying the water-soluble and water-insoluble components of B16F10 cells. The nanovaccine was then used to treat tumors in B16F10 melanoma-bearing mice.

[0093] (1) Lysis of cancer cells and collection of each fraction A fixed amount of B16F10 cells was harvested, the culture medium removed, and frozen at -20°C to -273°C. A fixed amount of ultrapure water was added, and the cells were subjected to three or more freeze-thaw cycles, optionally with sonication, to disrupt the lysed cells. After cell lysis, the lysate was centrifuged at 100 g or higher for at least one minute. The supernatant, i.e., the water-soluble components of the B16F10 cells, were collected. 8M urea was added to the resulting precipitate to dissolve the precipitate, and the water-insoluble components of the B16F10 cells were solubilized in an 8M urea solution. The water-soluble components from the cancer cell lysate and the original water-insoluble components dissolved in 8M urea were used as raw materials for the production of nanovaccines for cancer treatment.

[0094] (2) Manufacturing of nanovaccines In this example, the nanovaccine and blank nanoparticles used as a control were prepared using a double emulsion method with solvent evaporation. The molecular weight of the nanoparticle preparation material, PLGA, was 24 kDa to 38 kDa. The immunoadjuvant used was poly(I:C), which was distributed inside the nanoparticles and also adsorbed on the surface. The preparation method was as described above. Before loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanovaccine obtained was approximately 300 nm, and the Zeta potential of the nanovaccine surface was approximately -5 mV. Approximately 150 μg of protein or polypeptide fraction was loaded per mg of PLGA nanoparticles. The total amount of poly(I:C) immunoadjuvant used on the inside and outside of each mg of PLGA nanoparticles was approximately 0.01 mg, half each. The particle size of the blank nanoparticles was approximately 215 nm. In preparing the blank nanoparticles, pure water or 8 M urea containing the same amount of poly(I:C) was used to replace the corresponding water-soluble and water-insoluble components, respectively, and the outer surface of the blank nanoparticles was adsorbed with the same amount of poly(I:C) as the nanovaccine.

[0095] (3) Using nanovaccines for cancer treatment In this study, two different administration methods were investigated separately: the use of nanovaccine alone; and the combination of nanovaccine with PD-1 antibody. The control groups were PBS and PD-1 antibody + blank nanoparticles + cell lysate, respectively.

[0096] Six to eight-week-old female C57BL / 6 mice were selected as model mice to prepare melanoma-bearing mice.

[0097] The administration protocol for the group using only the nanovaccine was as follows: On day 0, 150,000 B16F10 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanoparticles carrying the water-soluble components of cancer cell lysate inside and on their surface, and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea inside and on their surface were subcutaneously injected, respectively.

[0098] The administration regimen for the nanovaccine and PD-1 antibody combination group was as follows: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells in the lower right dorsal region. On days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanoparticles carrying the aqueous components of cancer cell lysate on their interior and surface, and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea on their interior and surface, respectively, were subcutaneously injected. PD-1 antibody was intraperitoneally injected on days 3, 6, 9, and 14, with each mouse receiving a 10 mg / kg injection.

[0099] The PBS blank control group was administered subcutaneously to each mouse at day 0, with 150,000 B16F10 cells injected into the lower right back. On days 4, 7, 10, 15, and 20, 400 μL of PBS was injected subcutaneously.

[0100] PD-1 antibody + blank nanoparticles + cell lysate control group: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells in the lower right dorsal region. On days 4, 7, 10, 15, and 20, 4 mg of PLGA blank nanoparticles loaded with the same amount of the soluble cancer cell lysate component, the same amount of the original insoluble cancer cell lysate dissolved in 8 M urea, and the same amount of poly(I:C) but without any other cell lysate components were subcutaneously injected. To avoid free cell lysate adsorption to the surface of the blank nanoparticles, these three nanoparticles were administered separately and injected at different sites. PD-1 antibody was intraperitoneally injected on days 3, 6, 9, and 14, with each mouse receiving a 10 mg / kg injection.

[0101] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. For ethical reasons in animal experiments, the survival study of mice was limited to mice with tumor volumes of 2000 mm. 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0102] (4) Experimental results As shown in Figure 18, compared with the PBS blank control group, the tumor growth rate in mice in the blank nanoparticle + cell lysate + PD1 antibody control group was significantly slower (p<0.05), and the survival time of mice was significantly prolonged (p<0.05). Furthermore, compared with the PBS blank control group and the blank nanoparticle + cell lysate + PD1 antibody control group, the tumor growth rate in the nanovaccine alone group and the nanovaccine + PD1 antibody combination group was significantly slower (p<0.05), and the survival time of mice was significantly prolonged (p<0.05). Furthermore, 25% of mice in both treatment groups were cured, with tumors completely disappearing. When the nanovaccine and PD1 antibody combination regimen was used in this example, the synergistic effect of the two on the prolongation of mouse survival was not significant.

[0103] In conclusion, the nanovaccine carrying the water-soluble and water-insoluble components of cancer cells described in the present invention has a therapeutic effect on melanoma and can partially cure melanoma.

[0104] Example 2: Water-soluble cellular components of melanoma cancer cells are loaded inside and on the surface of nanoparticles for use in cancer treatment. In this example, we used mouse melanoma as a cancer model to explain how to prepare a nanovaccine carrying only the water-soluble portion of a cellular fraction and how to use the vaccine to treat melanoma.

[0105] Due to its excellent clinical efficacy, PD-1 antibodies have been approved for the treatment of many cancers. Therefore, in this embodiment, the applicant used the nanovaccine in combination with a PD-1 antibody to treat melanoma. In actual use, nanovaccines prepared solely from the original water-insoluble components of cell fractions can also be used for treatment. Furthermore, the specific administration time, frequency, and method can be adjusted according to the circumstances.

[0106] In this example, B16F10 mouse melanoma cells were used as a cancer cell model. First, B16F10 cells were dissolved to produce water-soluble and water-insoluble components of B16F10 cells. Next, a nanovaccine carrying the water-soluble components of B16F10 cells was produced by solvent evaporation using the organic polymer material PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant. The nanovaccine was then used to treat tumors in melanoma-bearing mice.

[0107] (1) Lysis of cancer cells and collection of each fraction A certain amount of B16F10 cells was collected, the medium was removed, and then frozen at -20°C to -273°C. A certain amount of ultrapure water was added, and the cells were subjected to three or more freeze-thaw cycles, optionally with ultrasonic waves, to disrupt the lysed cells. After cell lysis, the lysate was centrifuged at 100g or higher for 5 minutes, and the supernatant, i.e., the water-soluble components of B16F10 soluble in pure water, were collected. The water-soluble components derived from the cancer cell lysate obtained were used as a raw material for producing nanovaccines for cancer treatment.

[0108] (2) Manufacturing of nanovaccines In this example, the nanovaccine and blank nanoparticles used as controls were prepared using a double emulsion method with solvent evaporation. The nanoparticle material used was the organic polymer PLGA with a molecular weight of 24 kDa to 38 kDa. The immunoadjuvant used was poly(I:C), which was distributed inside the nanoparticles and adsorbed on the surface. The preparation method was as described above. Before loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the nanovaccine particle size was approximately 300 nm, and the average surface potential Zeta potential of the nanoparticles was approximately -5 mV. Each mg of PLGA nanoparticles carried 150 μg of protein or polypeptide fraction, and 0.01 mg of poly(I:C) immunoadjuvant was used on the inside and outside of each mg of PLGA nanoparticles, half each. The particle size of the blank nanoparticles was approximately 215 nm. In preparing the blank nanoparticles, pure water or 8 M urea containing the same amount of poly(I:C) was used to replace the corresponding water-soluble and water-insoluble components, respectively, and the outer surface of the blank nanoparticles was adsorbed with the same amount of poly(I:C) as the nanovaccine.

[0109] (3) Using nanovaccines for cancer treatment In this study, a nanovaccine containing only water-soluble components was used in combination with a PD-1 antibody to treat cancer.

[0110] Six to eight week old female C57BL / 6 mice were selected to generate melanoma-bearing mice.

[0111] The administration protocol for the nanovaccine and PD-1 antibody combination group was as follows: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells in the lower right dorsal region. On days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanovaccine nanoparticles loaded with the aqueous components of cancer cell lysate on their interior and surface, 200 μL of 2 mg PLGA blank nanoparticles loaded with an equal amount of poly(I:C), and the insoluble components of cell lysate dissolved in 8 M urea (containing an equal amount of poly(I:C)) were injected subcutaneously at three different subcutaneous sites. On days 3, 6, 9, and 14, PD-1 antibody was injected intraperitoneally at a dose of 10 mg / kg per mouse.

[0112] The PBS blank control group was administered subcutaneously to each mouse at day 0, with 150,000 B16F10 cells injected into the lower right back. 400 μL of PBS was injected subcutaneously on days 4, 7, 10, 15, and 20.

[0113] PD-1 antibody + blank nanoparticles + cell lysate control group: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells in the lower right dorsal region. On days 4, 7, 10, 15, and 20, 200 μL of the same amount of aqueous cancer cell lysate, 200 μL of the same amount of the original insoluble cancer cell lysate dissolved in 8 M urea, and 4 mg of PLGA blank nanoparticles loaded with the same amount of poly(I:C) but without the cell lysate component were subcutaneously injected. To avoid free cell lysate adsorption to the surface of the blank nanoparticles, these three nanoparticles were administered separately and injected at different sites. PD-1 antibody was intraperitoneally injected on days 3, 6, 9, and 14, with each mouse receiving a 10 mg / kg injection.

[0114] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2where v is the tumor volume, a is the tumor length, and b is the tumor width. For ethical reasons in animal experiments, the survival study of mice was limited to mice with tumor volumes of 2000 mm. 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0115] (4) Experimental results As shown in Figure 19, the tumor growth rate in mice in the blank nanoparticle + cell lysate + PD-1 antibody control group and the nanovaccine-treated group was significantly slower (p<0.05) and the survival time of mice was significantly prolonged (p<0.05) compared with the PBS blank control group. Furthermore, the tumor growth rate in mice in the nanovaccine-treated group was significantly slower (p<0.05) compared with the blank nanoparticle + cell lysate + PD-1 antibody control group. Furthermore, 12.5% ​​of mice in the nanovaccine-treated group were cured, with tumors completely disappearing. This demonstrates that the nanovaccine loaded with water-soluble components of cancer cells described in this invention is effective in treating melanoma and can partially cure melanoma.

[0116] Example 3: Melanoma tumor tissue lysate fraction loaded into and on nanoparticles for use in cancer treatment In this embodiment, a method for treating melanoma was described in which a nanovaccine carrying a melanoma tumor tissue lysate fraction was produced using mouse melanoma, and the vaccine was used.

[0117] In this embodiment, the applicant tested the effect of combining the nanovaccine with PD-1 antibody to treat melanoma, but in actual use, the nanovaccine alone may also be used to treat cancer.

[0118] In this example, a nanovaccine was prepared by loading a soluble fraction of mouse melanoma tumor tissue into the interior and surface of nanoparticles. First, a mouse melanoma tumor mass was removed and dissolved to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, using PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant, a nanovaccine was prepared by loading the water-soluble and water-insoluble components of the tumor tissue lysate using the solvent evaporation method. The nanovaccine was then used to treat tumors in melanoma tumor-bearing mice.

[0119] (1) Lysis of tumor tissue and collection of each fraction Each C57BL / 6 mouse was subcutaneously inoculated with 150,000 B16F10 melanoma cells into the back, and the inoculated tumors in each mouse were approximately 80 mm 3 , 200mm 3 , 400mm 3 , 1000mm 3 When the tumor tissues reached a volume of 1000mcg, the mice were sacrificed and tumor tissues were collected. Tumor tissues of the same size were cut and polished, passed through a cell strainer, and subjected to five cycles of freezing and thawing. After the tumor tissue cells were dissolved, the tumor mass tissue cell lysate was centrifuged at a speed exceeding 12,000 RPM for 5 minutes to obtain the supernatant, i.e., the water-soluble components of the tumor mass tissue that were soluble in pure water; the resulting precipitate was dissolved by adding an 8M urea aqueous solution, converting the original water-insoluble components that were insoluble in pure water into those that were soluble in 8M urea aqueous solution. The water-soluble components of the obtained tumor tissue lysate and the original water-insoluble components dissolved in 8M urea were Nwo It was a source of raw materials for producing nanovaccines for treatment.

[0120] (2) Manufacturing of nanovaccines In this example, the nanovaccine and blank nanoparticles used as a control were prepared using a double emulsion method with solvent evaporation. The molecular weight of the nanoparticle preparation material, PLGA, ranged from 24 kDa to 38 kDa. The immunoadjuvant used was poly(I:C), which was distributed inside the nanoparticles and also adsorbed onto their surfaces. The preparation method was as described above. Before loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the nanovaccine particle size was approximately 300 nm, and the average surface potential Zeta potential of the nanovaccine was approximately -5 mV. Each mg of PLGA nanoparticles carried 160 μg of protein or polypeptide fraction, and 0.01 mg of poly(I:C) immunoadjuvant was used on the inside and outside of each mg of PLGA nanoparticles, half each. The particle size of the blank nanoparticles was approximately 215 nm. In preparing the blank nanoparticles, pure water or 8 M urea containing the same amount of poly(I:C) was used to replace the corresponding water-soluble and water-insoluble components, respectively, and the surface of the blank nanoparticles was adsorbed with the same amount of poly(I:C) as the nanovaccine.

[0121] (3) Using nanovaccines for cancer treatment Six to eight week old female C57BL / 6 mice were selected to generate melanoma-bearing mice.

[0122] The nanovaccine group was administered the following protocol: On day 0, 150,000 B16F10 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanoparticles carrying the water-soluble components of tumor tissue lysate inside and on their surface, and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea inside and on their surface, were subcutaneously injected. Here, the tumor tissue lysate carried by the nanovaccine injected on days 4 and 7 was approximately 80 mm in size. 3The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected on day 10 was approximately 200 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected on day 15 was approximately 400 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected on day 20 was approximately 1000 mm in size. 3 The PD-1 antibody was intraperitoneally injected into each mouse on days 3, 6, 9, and 14, with the injection dose per mouse being 10 mg / kg.

[0123] The PBS blank control group was administered as follows: on day 0, 150,000 B16F10 cells were subcutaneously inoculated into the lower right back of each mouse, and 400 μL of PBS was injected subcutaneously on days 4, 7, 10, 15, and 20.

[0124] PD-1 antibody + blank nanoparticles + cell lysate control group: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells in the lower right dorsal region. On days 4, 7, 10, 15, and 20, the same amount of the soluble cancer cell lysate fraction, the same amount of the original insoluble cancer cell lysate fraction dissolved in 8 M urea, and 4 mg of PLGA blank nanoparticles loaded with the same amount of poly(I:C) but without the cell lysate fraction were subcutaneously injected. To avoid free cell lysate adsorption to the surface of the blank nanoparticles, these three nanoparticles were administered separately and injected at different sites. PD-1 antibody was intraperitoneally injected on days 3, 6, 9, and 14, with each mouse receiving a 10 mg / kg injection.

[0125] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. For ethical reasons in animal experiments, the survival study of mice was limited to mice with tumor volumes of 2000 mm. 3If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0126] (4) Experimental results As shown in Figure 20, compared with the PBS blank control group and the blank nanoparticles + tissue lysate + PD1 antibody control group, the tumor volume growth rate in the nanovaccine-treated group was significantly slower (p<0.05), and the survival time of mice was significantly prolonged (p<0.05). Furthermore, 25% of the mice in the nanovaccine-treated group were cured, with tumors completely disappearing. This indicates that the nanovaccine containing the water-soluble and water-insoluble components of tumor tissue lysate described in this invention has a therapeutic effect against melanoma and can partially cure melanoma.

[0127] Example 4: Whole cell fractions of melanoma cancer cells loaded inside and on nanoparticles for use in cancer prevention In this example, a method for preventing melanoma was described using a mouse melanoma cancer model, producing a nanovaccine carrying a whole cell fraction, and using the vaccine.

[0128] In this example, B16F10 mouse melanoma cells were used as a cancer cell model. First, B16F10 cells were lysed to prepare the water-soluble and water-insoluble components of B16F10 cells. Next, a nanovaccine carrying the water-soluble and water-insoluble components of B16F10 cells was prepared by solvent evaporation using PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant. The nanovaccine was then used to prevent melanoma.

[0129] (1) Lysis of cancer cells and collection of each fraction The method for lysing the cancer cells and the method for collecting each fraction were the same as in Example 1.

[0130] (2) Manufacturing of nanovaccines In this example, the nanovaccine and blank nanoparticles used as a control were prepared using a double emulsion method with solvent evaporation. The molecular weight of the nanoparticle preparation material, PLGA, was 24 kDa to 38 kDa. The immunoadjuvant used was poly(I:C), which was distributed inside the nanoparticles and also adsorbed on the surface. The preparation method was as described above. Before loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the particle size of the nanovaccine obtained was approximately 300 nm, and the average surface potential Zeta potential of the nanovaccine was approximately -5 mV. Each mg of PLGA nanoparticles was loaded with 150 μg of protein or polypeptide fraction, and 0.01 mg of poly(I:C) immunoadjuvant was used on the inside and outside of each mg of PLGA nanoparticles, half each. The particle size of the blank nanoparticles was approximately 215 nm. In the preparation of the blank nanoparticles, pure water containing poly(I:C) or 8 M urea was used to replace the corresponding water-soluble and water-insoluble components, respectively, and the surface of the blank nanoparticles was adsorbed with the same amount of poly(I:C) as the nanovaccine.

[0131] (3) Using nanovaccines to prevent cancer Six- to eight-week-old female C57BL / 6 mice were used to prepare melanoma-bearing mice. On days 42, 35, 28, 21, and 14 before inoculation with B16F10 cancer cells, 200 μL of a 2 mg PLGA nanovaccine containing the aqueous components of cancer cell lysate on its interior and surface, and 200 μL of a 2 mg PLGA nanovaccine containing the original water-insoluble components dissolved in 8 M urea on its interior and surface, were subcutaneously injected. 14 days after the final nanovaccine injection, each mouse was subcutaneously inoculated with 150,000 B16-F10 cells in the lower right dorsum. This day was designated day 0 of tumor cell inoculation.

[0132] In this experiment, the control group was administered the following protocol: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells into the lower right back.

[0133] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. For ethical reasons in animal experiments, in mouse survival experiments, mice were allowed to grow to a tumor volume of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0134] (4) Experimental results As shown in Figure 21, the tumor growth rate in the nanovaccine-vaccinated group was significantly slower (p<0.05) and the survival time of the mice was significantly longer (p<0.05) compared to the control group. Furthermore, tumors disappeared in approximately 35% of the mice after vaccination. This indicates that the nanovaccine carrying the water-soluble and water-insoluble components of the cancer cell lysate described in this invention has a preventive effect against melanoma and can prevent melanoma in some vaccine recipients.

[0135] Example 5: Melanoma tumor tissue lysate fraction loaded inside and on the surface of nanoparticles for use in cancer prevention In this example, a method for preventing melanoma was described using a mouse melanoma cancer model to produce a nanovaccine carrying a melanoma tumor tissue lysate fraction and using the vaccine.

[0136] In this example, a nanovaccine was prepared by loading a soluble fraction of mouse melanoma tumor tissue into the interior and surface of nanoparticles. Mouse melanoma tumor masses were first removed and lysed to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, using PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant, a nanovaccine was prepared by loading the water-soluble and water-insoluble components of the tumor tissue lysate using the solvent evaporation method. The nanovaccine was then used to treat tumors in B16F10 melanoma tumor-bearing mice.

[0137] (1) Lysis of tumor tissue and collection of each fraction The tumor tissue treatment method was the same as in Example 3. First, 150,000 B16F10 melanoma cells were subcutaneously inoculated into the back of each C57BL / 6 mouse, and the inoculated tumors of each mouse were approximately 80 mm 3 , 200mm 3 , 400mm 3 , 1000mm 3 When tumor tissue reached a volume of 1000 μg, mice were sacrificed and tumor tissue was collected. Tumor tissue of the same size was cut and polished, passed through a cell strainer, and subjected to freeze-thaw cycles at least three times. After tumor tissue cells were lysed, the tumor mass tissue cell lysate was centrifuged at 12,000 g for 5 minutes to obtain the supernatant, i.e., the water-soluble components of the tumor mass tissue that were soluble in pure water. The resulting precipitate was dissolved by adding 8M urea, converting the original water-insoluble components of B16F10 into those soluble in 8M urea solution. The resulting water-soluble and original water-insoluble components of the tumor tissue lysate were used as raw materials for the production of nanovaccines for melanoma prevention.

[0138] (2) Manufacturing of nanovaccines In this example, nanovaccines and blank nanoparticles (controls) were prepared using a double emulsion method with solvent evaporation. The nanoparticle material used was PLGA with a molecular weight of 24 kDa to 38 kDa. The immunoadjuvant used was poly(I:C), which was distributed inside the nanoparticles and also adsorbed onto their surfaces. The preparation method was as described above. Before loading the nanoparticles with the cell fraction and immunoadjuvant, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticles, the nanovaccine particle size was approximately 300 nm, and the average surface potential Zeta potential of the nanovaccine was approximately -5 mV. Each mg of PLGA nanoparticles carried 160 μg of protein or polypeptide fraction, and 0.01 mg of poly(I:C) immunoadjuvant was used on the inside and outside of each mg of PLGA nanoparticles, half each. The particle size of the blank nanoparticles was approximately 215 nm. In preparing the blank nanoparticles, pure water or 8 M urea containing the same amount of poly(I:C) was used to replace the corresponding water-soluble and water-insoluble components, respectively, and the same amount of poly(I:C) as the nanovaccine was adsorbed onto the surface of the blank nanoparticles.

[0139] (3) Using nanovaccines to prevent cancer Six to eight week old female C57BL / 6 mice were selected to prepare melanoma-bearing mice. On days 42, 35, 28, 21, and 14 before tumor cell inoculation, 200 μL of 2 mg PLGA nanoparticles carrying water-soluble components inside and on their surface and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea inside and on their surface were subcutaneously injected. Here, tumor tissue lysates carried by the nanovaccines injected on days 42 and 35 before tumor cell inoculation were approximately 80 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected 28 days before tumor cell inoculation was approximately 200 mm in size. 3The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected 21 days before tumor cell inoculation was approximately 400 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected 14 days before tumor cell inoculation was approximately 1000 mm in size. 3 Fourteen days after the final nanovaccine injection, each mouse was subcutaneously inoculated with 150,000 B16F10 cells into the lower right back, and this day was designated as day 0 of tumor cell inoculation.

[0140] In this experiment, the control group was administered the following protocol: On day 0, each mouse was subcutaneously inoculated with 150,000 B16F10 cells into the lower right back.

[0141] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. For ethical reasons in animal experiments, in mouse survival experiments, mice were allowed to grow to a tumor volume of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0142] (4) Experimental results As shown in Figure 22, compared with the PBS blank control group, the tumor growth rate in the nanovaccine-vaccinated group was significantly slower (p<0.05), and the survival time of the mice was significantly prolonged (p<0.05). Furthermore, approximately 35% of the tumors in the mice disappeared after vaccination. This indicates that the nanovaccine containing the water-soluble and water-insoluble components of tumor tissue lysate described in this invention has a preventive effect against melanoma and can prevent melanoma in some vaccine recipients.

[0143] Example 6: Whole cell fractions of breast cancer cells are loaded inside and on nanoparticles for use in cancer treatment. In this example, we used the treatment of breast cancer in mice to demonstrate how to prepare a nanovaccine loaded with a whole cell fraction and use the vaccine to treat breast cancer.

[0144] In this example, in addition to using the nanovaccine alone to treat breast cancer, the applicant also tested the combination of the nanovaccine with PD-1 antibody. In actual use, the specific administration time, administration frequency, and administration method can be adjusted according to the situation.

[0145] In this example, 4T1 mouse triple-negative breast cancer cells were used as a cancer cell model. First, 4T1 cells were lysed to produce water-soluble and water-insoluble components of the 4T1 cells. Next, a nanovaccine was prepared by solvent evaporation using PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant, carrying the water-soluble and water-insoluble components of the 4T1 cells. The nanovaccine was then used to treat tumors in 4T1 breast cancer-bearing mice.

[0146] (1) Lysis of cancer cells and collection of each fraction In this example, the methods for lysing cancer cells, recovering the lysate, and solubilizing the cells were the same as in Example 1, except that B16F10 cells were replaced with 4T1 cells.

[0147] (2) Manufacturing of nanovaccines The method for producing the nanovaccine and the materials used in this example were all the same as those in Example 1, except that B16F10 cells were replaced with 4T1 cells.

[0148] (3) Using nanovaccines for cancer treatment Six to eight week old female BALB / c mice were selected to produce 4T1 tumor-bearing mice.

[0149] The administration protocol for the nanovaccine group was as follows: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right back region, and on days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanoparticles carrying the water-soluble components of cancer cell lysate inside and on their surface, and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea inside and on their surface were subcutaneously injected, respectively.

[0150] The administration regimen for the nanovaccine and PD-1 antibody combination group was as follows: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right back region. On days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanoparticles with water-soluble components loaded on the interior and surface, and 200 μL of 2 mg PLGA nanoparticles with the original water-insoluble components dissolved in 8 M urea loaded on the interior and surface, respectively, were subcutaneously injected. On days 3, 6, 9, and 14, PD-1 antibody was intraperitoneally injected at a dose of 10 mg / kg per mouse.

[0151] The PBS blank control group was administered as follows: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and 400 μL of PBS was injected subcutaneously on days 4, 7, 10, 15, and 20.

[0152] PD-1 antibody + blank nanoparticles + cell lysate control group: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right dorsum. On days 4, 7, 10, 15, and 20, the aqueous fraction of cancer cell lysate, the original insoluble fraction of cancer cell lysate dissolved in 8 M urea, and 4 mg of PLGA blank nanoparticles loaded with the same amount of poly(I:C) but without the cell lysate component were subcutaneously injected. To avoid free cell lysate adsorption to the surface of the blank nanoparticles, these three nanoparticles were administered separately and injected at different sites. PD-1 antibody was intraperitoneally injected on days 3, 6, 9, and 14, with each mouse receiving a 10 mg / kg injection.

[0153] The administration method for the group receiving the nanovaccine containing only water-soluble components in combination with the PD-1 antibody was as follows: On day 0, a small incision was placed in the lower right back of each mouse. 400,000 4T1 cells The vaccine was subcutaneously administered to mice. On days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanovaccine nanoparticles containing the aqueous components of cancer cell lysate, 200 μL of 2 mg PLGA blank nanoparticles containing an equal amount of poly(I:C) alone, and 200 μL of the insoluble components dissolved in 8 M urea were subcutaneously injected at three different subcutaneous sites. On days 3, 6, 9, and 14, PD-1 antibody was intraperitoneally injected at a dose of 10 mg / kg per mouse.

[0154] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. In mouse survival studies, mice with tumor volumes of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0155] (4) Experimental results As shown in Figure 23, compared with the PBS blank control group and the blank nanoparticles + cell lysate + PD1 antibody control group, the tumor growth rate in the nanovaccine administration group was significantly slower (p<0.05) and the survival time of the mice was significantly prolonged (p<0.05), demonstrating that the nanovaccine carrying the water-soluble and water-insoluble components of cancer cells described in the present invention has a therapeutic effect against breast cancer.

[0156] When the combined administration method of nanovaccine and PD1 antibody in this example was adopted, the two did not have a significant synergistic effect on inhibiting tumor growth rate and extending the survival time of mice.

[0157] Comparing the nanovaccine containing only water-soluble components + PD1 antibody group with the blank nanoparticle + cell lysate + PD1 antibody control group, there was no significant difference in tumor growth inhibition and mouse survival time, indicating that the nanovaccine containing only water-soluble components does not have a significant therapeutic effect in the treatment of breast cancer.

[0158] Example 7: Breast cancer tumor tissue lysate fraction loaded inside and on the surface of nanoparticles for use in cancer treatment In this example, we use mouse breast cancer as a cancer model to prepare a nanovaccine carrying a breast cancer tumor tissue lysate fraction, and describe a method for treating breast cancer using the vaccine. In actual use, the specific administration time, frequency, and administration method can be adjusted according to the situation.

[0159] In this example, a nanovaccine was prepared by loading the soluble fraction of mouse breast cancer tumor tissue into the interior and surface of nanoparticles. Mouse breast cancer tumor masses were first removed and lysed to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, using PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant, a nanovaccine was prepared by loading the water-soluble and water-insoluble components of the tumor tissue lysate using the solvent evaporation method. The nanovaccine was then used to treat tumors in breast cancer tumor-bearing mice.

[0160] (1) Lysis of tumor tissue and collection of each fraction The methods for lysing cancer cells and recovering and solubilizing the lysate in this example were the same as those in Example 3, except that melanoma tumor mass cells were replaced with breast cancer tumor masses.

[0161] (2) Manufacturing of nanovaccines The nanovaccine manufacturing method and materials used in this example were the same as those in Example 3, except that melanoma tumor tissue was replaced with breast cancer tumor tissue.

[0162] (3) Using nanovaccines for cancer treatment Six to eight week old female BALB / c mice were selected to generate 4T1 tumor-bearing mice.

[0163] The nanovaccine treatment group was administered as follows: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanoparticles carrying water-soluble components inside and on the surface and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea inside and on the surface were subcutaneously injected. Here, the tumor tissue lysates carried by the nanovaccine injected on days 4 and 7 were approximately 80 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected on day 10 was approximately 200 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected on day 15 was approximately 400 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected on day 20 was approximately 1000 mm in size. 3 It was derived from a tumor mass.

[0164] The PBS blank control group was administered as follows: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and 400 μL of PBS was injected subcutaneously on days 4, 7, 10, 15, and 20.

[0165] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. In mouse survival studies, mice with tumor volumes of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0166] (4) Experimental results As shown in Figure 24, the tumor growth rate in the nanovaccine-administered group was significantly slower (p<0.05) and the survival time of the mice was significantly prolonged (p<0.05) compared with the PBS blank control group. This indicates that the nanovaccine carrying the water-soluble and water-insoluble components of tumor tissue described in the present invention has a therapeutic effect against breast cancer.

[0167] Example 8: Whole cell fractions of breast cancer cells are loaded into and onto microparticles for use in cancer treatment. In this example, using mouse breast cancer as a cancer model, the production of a microvaccine carrying a whole cell fraction and a method for treating breast cancer using the vaccine were described.

[0168] In this example, 4T1 mouse triple-negative breast cancer cells were used as a cancer cell model. 4T1 cells were first lysed to prepare the water-soluble and water-insoluble components of the 4T1 cells. Next, a microvaccine containing the water-soluble and water-insoluble components of the 4T1 cells was prepared by solvent evaporation using PLGA as the microparticle scaffold and poly(I:C) as the immunoadjuvant. The microvaccine was then used to treat tumors in 4T1 breast cancer tumor-bearing mice.

[0169] (1) Lysis of cancer cells and collection of each fraction The methods for lysing cancer cells and recovering and solubilizing the lysate in this example were the same as those in Example 1, except that B16F10 cells were replaced with 4T1 cells.

[0170] (2) Production of microvaccines The manufacturing method and materials of the microvaccine and blank microparticles in this example are similar to those in Example 1, except that the sonication time of colostrum and composite milk in the microparticle manufacturing by the double emulsion method is shorter. The average particle diameter of the manufactured microvaccine is about 2 μm, the surface zeta potential of the microparticles is -4 mV, the protein or polypeptide fraction supported on the inside and outside of the PLGA clonal particles per 1 mg is 200 μg, and the protein or polypeptide fraction supported on the inside and outside of the PLGA clonal particles per 1 mg is 200 μg. micro The total amount of poly(I:C) immunoadjuvant used inside and outside the particles was 0.01 mg, half inside and half outside.

[0171] (3) Using microvaccines for cancer treatment Six to eight week old female BALB / c mice were selected to generate 4T1 tumor-bearing mice.

[0172] The microvaccine treatment group was administered as follows: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right back region, and on days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA microparticles carrying water-soluble cancer components on their interior and surface, and 200 μL of 2 mg PLGA microparticles carrying the original water-insoluble components dissolved in 8 M urea on their interior and surface, were subcutaneously injected.

[0173] The PBS blank control group was administered as follows: on day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and 400 μL of PBS was injected subcutaneously on days 4, 7, 10, 15, and 20.

[0174] Blank microparticle + cell lysate control group: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right back. On days 4, 7, 10, 15, and 20, the same amount of the aqueous component of the cancer cell lysate, the same amount of the original water-insoluble component of the cancer cell lysate dissolved in 8 M urea, and 4 mg of PLGA blank microparticles loaded with the same amount of poly(I:C) but without the cell lysate component were subcutaneously injected. To avoid free cell lysate adsorption on the surface of the blank microparticles, these three components had to be administered separately and injected at different sites.

[0175] During the experiment, the size of the tumor volume of the mice was recorded every three days. The tumor volume was calculated using the formula v = 0.52 * a * b 2where v is the tumor volume, a is the tumor length, and b is the tumor width. In mouse survival studies, mice with tumor volumes of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0176] (4) Experimental results As shown in Figure 25, the tumor growth rate in the microvaccine-treated group was significantly slower (p<0.05) and the survival time of the mice was significantly longer (p<0.05) compared with the PBS blank control group and the blank microparticle control group, demonstrating that the microvaccine carrying the water-soluble and water-insoluble components of cancer cells described in the present invention has a therapeutic effect against breast cancer.

[0177] Example 9: Breast cancer tumor tissue lysate fraction loaded inside and on the surface of nanoparticles for use in cancer prevention In this example, a method for preventing breast cancer was described in which a nanovaccine carrying breast cancer tumor tissue lysate components was produced using mouse breast cancer as a cancer model, and the vaccine was used.

[0178] In this example, a nanovaccine was prepared by loading the soluble fraction of mouse breast cancer tumor tissue into the interior and surface of nanoparticles. Mouse breast cancer tumor masses were first removed and lysed to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, using PLGA as the nanoparticle scaffold and poly(I:C) as the immunoadjuvant, a nanovaccine was prepared by loading the water-soluble and water-insoluble components of the tumor tissue lysate using the solvent evaporation method. The nanovaccine was then used to treat tumors in breast cancer tumor-bearing mice.

[0179] (1) Lysis of tumor tissue and collection of each fraction The methods for lysing tumor tissue and recovering and solubilizing the lysate in this example were the same as those in Example 3, except that the melanoma tumor mass was replaced with a breast cancer tumor mass.

[0180] (2) Manufacturing of nanovaccines The nanovaccine manufacturing method and materials used in this example were the same as in Example 3, except that melanoma tumor masses were replaced with breast cancer tumor masses. Before loading the nanoparticles with the cell fraction and immunoadjuvant, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the particle size of the resulting nanovaccine was approximately 300 nm. Each mg of PLGA nanoparticles loaded 150 μg of protein or polypeptide. Each mg of PLGA nanoparticles contained 0.01 mg of poly(I:C) immunoadjuvant, half on the inside and half on the outside. The zeta potential of the resulting nanovaccine surface was -5 mV.

[0181] (3) Using nanovaccines to prevent cancer Six to eight week old female BALB / c mice were selected to produce 4T1 tumor-bearing mice.

[0182] Nanovaccine prevention group regimen: 200 μL of 2 mg PLGA nanoparticles carrying water-soluble components on the inside and surface and 200 μL of 2 mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8 M urea on the inside and surface were subcutaneously injected on days 42, 35, 28, 21, and 14 before tumor cell inoculation. Here, the tumor tissue lysates carried by the nanovaccine injected on days 42 and 35 before tumor inoculation were approximately 80 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected 28 days before inoculation of the mouse tumor was approximately 200 mm in size. 3 The tumor tissue lysate derived from the tumor mass and loaded by the nanovaccine injected 21 days before tumor inoculation was approximately 400 mm in size. 3 The tumor tissue lysate derived from the tumor mass and carried by the nanovaccine injected 14 days before tumor inoculation was approximately 1000 mm in size. 3 Fourteen days after the final vaccine injection, each mouse was subcutaneously inoculated with 400,000 4T1 cells into the lower right back, and this day was designated as day 0.

[0183] The control group was treated as follows: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells into the lower right back.

[0184] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0185] (4) Experimental results As shown in Figure 26, the tumor volume growth rate in the nanovaccine-prevented group was significantly slower (p<0.05) and the survival time of the mice was significantly longer (p<0.05) compared to the control group. This indicates that the nanovaccine containing the water-soluble and water-insoluble components of breast cancer tumor tissue lysate described in this invention has a preventive effect against melanoma.

[0186] Example 10: Whole cell fractions of breast cancer cells are loaded inside and on the surface of nanoparticles and used as a nanovaccine with BCG as an immunoadjuvant for cancer treatment. In this example, we used mouse breast cancer as a cancer model, produced a nanovaccine carrying a whole cell fraction using BCG as an immune adjuvant, and explained a method for treating breast cancer using this vaccine.

[0187] In this example, 4T1 mouse triple-negative breast cancer cells were used as a cancer cell model. First, 4T1 cells were lysed to extract the water-soluble and water-insoluble components of the 4T1 cells. Next, a nanovaccine was prepared by solvent evaporation using PLGA as the nanoparticle scaffold and BCG as the immunoadjuvant, carrying the water-soluble and water-insoluble components of the 4T1 cells. The nanovaccine was then used to treat tumors in 4T1 breast cancer-bearing mice.

[0188] (1) Lysis of cancer cells and collection of each fraction The methods for lysing cancer cells and recovering and solubilizing the lysate in this example were the same as those in Example 1, except that B16F10 cells were replaced with 4T1 cells.

[0189] (2) BCG dissolution and collection of each fraction The methods for dissolving BCG and recovering and solubilizing the lysate in this example were the same as the method for dissolving cancer cells in Example 1, except that the cancer cells were replaced with BCG.

[0190] (3) Manufacturing of nanovaccines The nanovaccine manufacturing method and materials used in this example were the same as those in Example 6. In this example, the immunoadjuvant loaded inside the nanovaccine was replaced from poly(I:C) with the water-soluble or water-insoluble components of BCG lysate; the immunoadjuvant adsorbed on the surface of the nanovaccine was undissolved BCG adjuvant. Before loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanoparticles was approximately 250 nm. After loading the cell fraction and immunoadjuvant onto the nanoparticle surface, the average particle size of the nanovaccine prepared was approximately 310 nm. The amount of protein and polypeptide loaded on the inside and outside of the PLGA microparticles per mg was 150 μg. The amount of BCG immunoadjuvant used inside and outside of the PLGA nanoparticles per mg was 0.1 mg, half each.

[0191] (4) Using nanovaccines for cancer treatment Six to eight week old female BALB / c mice were selected to produce 4T1 tumor-bearing mice.

[0192] The nanovaccine and PD-1 antibody combination regimen was as follows: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right back region. On days 4, 7, 10, 15, and 20, 200 μL of 2 mg PLGA nanovaccine with water-soluble components inside and on its surface, and 200 μL of 2 mg PLGA nanovaccine with the original water-insoluble components dissolved in 8 M urea inside and on its surface, were subcutaneously injected. On days 3, 6, 9, and 14, PD-1 antibody was intraperitoneally injected at a dose of 10 mg / kg per mouse.

[0193] In this experiment, the PBS blank control group was administered as follows: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and 400 μL of PBS was injected subcutaneously on days 4, 7, 10, 15, and 20.

[0194] PD-1 antibody + blank nanoparticle + cell lysate control group: On day 0, each mouse was subcutaneously inoculated with 400,000 4T1 cells in the lower right dorsal region. On days 4, 7, 10, 15, and 20, the same amount of the aqueous fraction of cancer cell lysate, the same amount of the original insoluble fraction of cancer cell lysate dissolved in 8 M urea, and 4 mg of PLGA blank nanoparticles containing the same amount of BCG but without the cell lysate component were subcutaneously injected. To avoid free cell lysate adsorption to the surface of the blank nanoparticles, these three nanoparticles were administered separately and injected at different sites. PD-1 antibody was intraperitoneally injected on days 3, 6, 9, and 14, with each mouse receiving a 10 mg / kg injection.

[0195] In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 6. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. In mouse survival studies, mice with tumor volumes of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0196] (4) Experimental results As shown in Figure 27, the tumor growth rate in the BCG-adjuvanted nanovaccine group was significantly slower (p<0.05) and the survival time of mice was significantly longer (p<0.05) compared with the PBS blank control group and the PD-1 antibody + blank nanoparticles + cell lysate control group. This indicates that the nanovaccine carrying the whole cell fraction of cancer cells described in this invention has a therapeutic effect against breast cancer when used with BCG as an adjuvant.

[0197] Example 11: Tumor tissue fractions are dissolved in 6 M guanidine hydrochloride, and the resulting solution is loaded onto the interior and surface of microparticles for use in cancer treatment. In this example, we used mouse breast cancer as a cancer model, lysing the whole cell fraction using 6M guanidine hydrochloride, and preparing a microvaccine containing the whole cell fraction to treat breast cancer. In this example, 4T1 mouse triple-negative breast cancer cells were used as a cancer cell model. First, tumor tissue cells were inactivated and denatured, and then the tumor tissue was lysed with 6M guanidine hydrochloride to lyse the whole cell fraction. Next, using PLGA as a microparticle scaffold and CpG as an immunoadjuvant, a microvaccine containing the whole cell fraction of tumor tissue was prepared using the solvent evaporation method. The microvaccine was then used to treat tumors in 4T1 breast cancer tumor-bearing mice.

[0198] (1) Lysis of cancer cells and collection of each fraction 400,000 4T1 cells were subcutaneously inoculated into the back of each BALB / c mouse, and the tumors inoculated into each mouse were approximately 80 mm 3 , 200mm 3 , 400mm 3 , 1000mm 3When the tumor tissue reached a volume of 1000, the mice were sacrificed and the tumor tissue was removed. Tumor tissues of the same size were cut, polished, and filtered through a cell strainer to collect the filtered tumor tissue cells. The resulting tumor tissue cells were inactivated and denatured by ultraviolet light and high-temperature heating, respectively, and then lysed using an appropriate amount of 6M guanidine hydrochloride to obtain a tissue lysate, providing a source of material for producing microvaccines.

[0199] (2) Production of microvaccines The manufacturing methods and materials used for the microvaccine and blank microparticles in this example were similar to those in Example 8, except that CpG was used as the immunoadjuvant. The average particle size of the prepared microvaccine was approximately 2.5 μm, and the surface Zeta potential of the microparticles was approximately -4 mV. The protein and polypeptide fractions loaded on the inside and outside of the PLGA microparticles per mg were 210 μg, and the PLGA per mg was 1.5 μg. micro The total amount of CpG immunoadjuvant used inside and outside the particles was 0.01 mg, half inside and half outside.

[0200] (3) Using microvaccines for cancer treatment Six to eight week old female BALB / c mice were selected to generate 4T1 tumor-bearing mice.

[0201] The microvaccine treatment group protocol was as follows: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 100 μL of 2 mg of PLGA microvaccine containing tumor tissue whole cell fraction was subcutaneously injected into the interior and exterior of the mouse. micro Vaccine-loaded tumor tissue lysates were approximately 80 mm in size. 3 derived from tumor mass and injected on day 10 micro Vaccine-loaded tumor tissue lysates were approximately 200 mm in size. 3 derived from tumor mass and injected on day 15 microVaccine-loaded tumor tissue lysates were approximately 400 mm in size. 3 derived from tumor mass and injected on day 20 micro Vaccine-loaded tumor tissue lysates were approximately 1000 mm in size. 3 It was derived from a tumor mass.

[0202] The PBS blank control group was as follows: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and 100 μL of PBS was subcutaneously injected on days 4, 7, 10, 15, and 20.

[0203] Blank microparticle + cell lysate control group: On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, the same amount of tumor tissue lysate and 2 mg of PLGA blank microparticles loaded with the same amount of CpG but without the cell lysate component were subcutaneously injected. micro To avoid adsorption of free cell lysate onto the particle surface, the two had to be administered separately and injected at different sites.

[0204] During the experiment, the size of the tumor volume of the mice was recorded every three days. The tumor volume was calculated using the formula v = 0.52 * a * b 2 where v is the tumor volume, a is the tumor length, and b is the tumor width. In mouse survival studies, mice with tumor volumes of 2000 mm 3 If the blood glucose level exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0205] (4) Experimental results As shown in Figure 28, the tumor growth rate in the microvaccine-treated group was significantly slower (p<0.05) and the survival time of mice was significantly longer (p<0.05) compared with the PBS blank control group and the blank microparticle control group, demonstrating that the microvaccine loaded with the whole cell fraction of tumor tissue described in the present invention has a therapeutic effect against breast cancer. [Brief explanation of the drawings]

[0206] [Figure 1] Figure 1 is a schematic diagram of the manufacturing process and field of use of the vaccine described in the present invention, where a: is a schematic diagram of collecting and manufacturing nanovaccines or microvaccines from water-soluble and water-insoluble components, respectively, and b: is a schematic diagram of dissolving the whole cell fraction using a solubilizing solution containing a solubilizing agent and manufacturing nanovaccines or microvaccines. [Figure 2]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 3]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 4]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 5]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 6]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 7]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 8]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 9]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 10]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 11]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 12]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 13]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 14]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 15]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 16]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 17]Schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, where 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immunopotentiating adjuvant, 4: nanoparticles or microparticles, and 5: the inner core of the nanoparticle. In Figures 2 to 5, the immunopotentiating adjuvant is contained on the surface and inside of the nanoparticles or microparticles. In Figures 6 to 9, the immunopotentiating adjuvant is distributed only inside the nanoparticles or microparticles. In Figures 10 to 13, the nanoparticles or microparticles contain the immunopotentiating adjuvant only on the outer surface. In Figures 14 to 17, the immunopotentiating adjuvant is not contained either inside or on the outer surface of the nanoparticles or microparticles. In Figures 2, 6, 10, and 14, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, they do not form a distinct inner core. In Figures 3, 7, 11, and 15, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, a single inner core portion is formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 4, 8, 12, and 16, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, multiple inner core portions are formed. The inner core is generated during the manufacturing process or formed using a polymer, inorganic salt, or the like. In Figures 5, 9, 13, and 17, when the water-soluble or water-insoluble components of the cell or tissue fraction carried by the nanoparticles or microparticles are distributed inside the nanoparticles or microparticles, the water-soluble or water-insoluble components are located in the outer layer of the formed inner core.a: Water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; b: Water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles; c: Water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-soluble components of the cell or tissue fraction are carried on the surface of both; d: Water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and water-insoluble components of the cell or tissue fraction are carried on the surface of both; e: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles; f: Nanoparticles g: Water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; h: Only the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle; i: Only the water-soluble components of the cell or tissue fraction are carried inside the nanoparticle or microparticle, and only the water-soluble components of the cell or tissue fraction are carried on the surface of the nanoparticle or microparticle. [Figure 18]These are experimental results of the effects of the nanovaccines in Examples 1 to 3 on the tumor growth rate and survival time of mice when used to treat melanoma; a, Experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b, Experiment on the effect of nanovaccine treatment on the survival time of tumor-inoculated mice (n≧8), each data point is the mean ± standard error (mean ± SEM); The significant difference in the tumor growth inhibition experiment in Figure a was analyzed using ANOVA, and the significant difference in Figure b was analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference between the group and the PBS blank control group at p<0.05; ☆ indicates a significant difference between the group and the blank nanoparticles + cell lysate + PD-1 antibody control group at p<0.05. [Figure 19] These are experimental results of the effects of the nanovaccines in Examples 1 to 3 on the tumor growth rate and survival time of mice when used to treat melanoma; a, Experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b, Experiment on the effect of nanovaccine treatment on the survival time of tumor-inoculated mice (n≧8), each data point is the mean ± standard error (mean ± SEM); The significant difference in the tumor growth inhibition experiment in Figure a was analyzed using ANOVA, and the significant difference in Figure b was analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference between the group and the PBS blank control group at p<0.05; ☆ indicates a significant difference between the group and the blank nanoparticles + cell lysate + PD-1 antibody control group at p<0.05. [Figure 20]These are experimental results of the effects of the nanovaccines in Examples 1 to 3 on the tumor growth rate and survival time of mice when used to treat melanoma; a, Experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b, Experiment on the effect of nanovaccine treatment on the survival time of tumor-inoculated mice (n≧8), each data point is the mean ± standard error (mean ± SEM); The significant difference in the tumor growth inhibition experiment in Figure a was analyzed using ANOVA, and the significant difference in Figure b was analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference between the group and the PBS blank control group at p<0.05; ☆ indicates a significant difference between the group and the blank nanoparticles + cell lysate + PD-1 antibody control group at p<0.05. [Figure 21] These are experimental results of the effects of the nanovaccines in Examples 1 to 3 on the tumor growth rate and survival time of mice when used to treat melanoma; a, Experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b, Experiment on the effect of nanovaccine treatment on the survival time of tumor-inoculated mice (n≧8), each data point is the mean ± standard error (mean ± SEM); The significant difference in the tumor growth inhibition experiment in Figure a was analyzed using ANOVA, and the significant difference in Figure b was analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference between the group and the PBS blank control group at p<0.05; ☆ indicates a significant difference between the group and the blank nanoparticles + cell lysate + PD-1 antibody control group at p<0.05. [Figure 22]Figure 1 shows experimental results of the effects of nanovaccines in Examples 4 and 5 on tumor growth rate and survival time in mice when used to prevent melanoma; a) Experiment on the inhibitory effect of nanovaccine treatment on tumor growth rate (n≧8); b) Experiment on the effect of nanovaccine treatment on survival time of tumor-inoculated mice (n≧8). Each data point is the mean±SEM. The significance of the tumor growth inhibition experiment in Figure a was analyzed using ANOVA, and the significance of the significance of the experiment in Figure b was analyzed using Kaplan-Meier and log-rank tests; * indicates a significant difference (p<0.05) between the group and the PBS blank control group. [Figure 23] Figure 1 shows experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11 on tumor growth rate and survival time in mice when used for breast cancer prevention or treatment; a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7); b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Data points are mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group; ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group. [Figure 24]Figure 1 shows experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11 on tumor growth rate and survival time in mice when used for breast cancer prevention or treatment; a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7); b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Data points are mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group; ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group. [Figure 25] Figure 1 shows experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11 on tumor growth rate and survival time in mice when used for breast cancer prevention or treatment; a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7); b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Data points are mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group; ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group. [Figure 26]Figure 1 shows experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11 on tumor growth rate and survival time in mice when used for breast cancer prevention or treatment; a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7); b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Data points are mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group; ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group. [Figure 27] Figure 1 shows experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11 on tumor growth rate and survival time in mice when used for breast cancer prevention or treatment; a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7); b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Data points are mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group; ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group. [Figure 28]Figure 1 shows experimental results of the effects of nanovaccines or microvaccines in Examples 6 to 11 on tumor growth rate and survival time in mice when used for breast cancer prevention or treatment; a) Experimental study of the tumor growth inhibitory effect of nanovaccines or microvaccines on tumor growth rate (n≧7); b) Experimental study of the effect of nanovaccines or microvaccines on survival time in tumor-inoculated mice (n≧7). Data points are mean±SEM. Significant differences in the tumor growth inhibition experiments in Figure a were analyzed using ANOVA, and significant differences in Figure b were analyzed using Kaplan-Meier and log-rank tests. * indicates a significant difference (p<0.05) between the corresponding group and the PBS blank control group; ☆ indicates a significant difference (p<0.05) between the corresponding group and the blank nanoparticles + cell lysate + PD-1 antibody control group.

Claims

1. The present invention comprises nanoscale-sized particles or microscale-sized particles and a whole cell fraction supported on the particles, wherein the whole cell fraction is a fraction comprising the following (A) and (B): Whole cell fraction delivery system for cancer prevention and / or treatment: (A) A water-soluble fraction, which is the supernatant obtained by lysing cancer cells in pure water or an aqueous solution containing no solubilizing agent and centrifuging the lysed cancer cells: (B) A water-insoluble fraction obtained by solubilizing the precipitate after centrifugation in (A) by adding a urea solution or a guanidine hydrochloride solution.

2. A delivery system as described in claim 1, wherein the water-soluble fraction and the water-insoluble fraction of whole cells are carried separately or together inside a particle and / or carried separately or together on the surface of a particle.

3. The delivery system of claim 2 , wherein the particle interior and / or surface further comprises an immune-enhancing adjuvant.

4. The delivery system of claim 3, wherein the immune-enhancing adjuvant is at least one of a microbial-derived immune enhancer, a product of the human or animal immune system, an innate immune agonist, an adaptive immune agonist, a chemically synthesized drug, a fungal polysaccharide, and a traditional Chinese medicine.

5. The immune-enhancing adjuvant may be a pattern recognition receptor agonist, BCG, BCG cell wall skeleton, BCG methanol extract residue, BCG muramyl dipeptide, Mycobacterium phlei, thymosin, polyactin A, mineral oil, virus-like particles, immune-enhancing reconstituted influenza virus, cholera enterotoxin, saponin and its derivatives, resiquimod, new bovine liver active peptide, miquimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly 4. The delivery system of claim 3, wherein the active ingredient is at least one of ICLC, Corynebacterium parvum, a Streptococcus pyogenes 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, and astragalus.

6. 10. The delivery system of claim 1, wherein the nanoscale-sized particles have a diameter of 1 nm to 1000 nm; the microscale-sized particles have a diameter of 1 μm to 1000 μm; or the surface of the nanoscale-sized particles or microscale-sized particles is electrically neutral, negatively charged, or positively charged.

7. The delivery system of claim 1 , wherein the nanoscale-sized particles or the microscale-sized particles are manufactured from an organic synthetic polymer material, a natural polymer material, or an inorganic material.

8. 8. The delivery system of claim 7, wherein the organic synthetic polymer material is PLGA, PLA, PGA, PEG, PCL, poloxamer, PVA, PVP, PEI, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, or synthetic polypeptide; the natural polymer material is lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membrane component, starch, sugar, or polypeptide; and the inorganic material is diiron trioxide, triiron tetroxide, calcium carbonate, or calcium phosphate.

9. 10. The delivery system of claim 1, wherein the shape is spherical, elliptical, barrel-shaped, polygonal, rod-shaped, sheet-shaped, wire-shaped, worm-shaped, square, triangular, butterfly-shaped, or disc-shaped.

10. Use of the whole cell fraction delivery system according to any one of claims 1 to 9 in the manufacture of a vaccine for the prevention and / or treatment of cancer.

11. A pharmaceutical composition or vaccine for the prevention and / or treatment of cancer, comprising a delivery system according to any one of claims 1 to 9.

12. A delivery system according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 11 for use in the manufacture of a vaccine for the prevention and / or treatment of cancer.

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