Targeted delivery system carrying whole cell fractions and uses thereof

Abstract

A targeted delivery system carrying a whole cell fraction, which relates to the technical field of immunotherapy, is described. The targeted delivery system is a nanoscale or microscale particle having a targeting head on its surface. The particle is loaded with a whole cell fraction of cancer cells or cancer tissues, the whole cell fraction being the water-soluble and water-insoluble components of all cells in the cell or tissue, the water-insoluble components being soluble with a solubilizing agent. The targeting head binds to molecules on the surface of specific cells or tissues, thereby helping the particle enter the cell or tissue. The targeted delivery system solubilizes the water-insoluble components using a specific solubilizing agent, making it soluble in aqueous solution. This allows the preparation of a cancer vaccine by combining the water-soluble components in the cancer cells or tissues with the whole cell antigens of the water-insoluble components. At the same time, the system is equipped with a targeting head capable of targeting antigen-presenting cells, thereby improving the efficiency of phagocytosis by antigen-presenting cells in an active targeting manner, further enhancing the efficacy of cancer prevention or treatment.

Description

Detailed Description of the Invention

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on October 23, 2020, bearing application number 202011146241.9 and entitled "Targeted delivery system carrying whole cell fraction and use thereof," the entire contents of which are incorporated herein by reference. [Technical field]

[0002] The present invention relates to the technical field of immunotherapy, in particular to a whole cell fraction-loaded targeted delivery system and uses thereof.

[0003] [Background technology] Immunity is a physiological function of the human body, which uses this function to distinguish between "self" and "non-self" components, destroy and reject antigenic substances (such as viruses and bacteria) that have invaded the human body, or damaged cells and tumor cells produced by the human body itself, and maintain the health of the human body. In recent years, immunological technology has developed extremely rapidly, especially 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 suppressing the occurrence and progression of cancer.

[0004] In recent years, cancer immunotherapy has developed rapidly, and cancer vaccines are one of the important methods for cancer immunotherapy and prevention. An effective cancer vaccine needs to carry cancer-specific antigens, and these carried antigens can be efficiently delivered to antigen-presenting cells to activate the body's immune system to recognize and attack cancer cells. Currently, scientists identify cancer-specific or cancer-related antigen polypeptides from tumor cell analysis of cancer patients and artificially synthesize them in vitro to produce cancer vaccines for cancer treatment. The method has shown a certain efficacy in clinical trials of cancer patients. However, the number of cancer antigens that can be discovered by such methods is limited, it is time-consuming and labor-intensive, and expensive, and the obtained polypeptide vaccines do not have the ability to target antigen-presenting cells.

[0005] To function as a vaccine, antigens must first be delivered to antigen-presenting cells, which then process the cancer antigens and present them on their surface, where they interact with T cells to activate immune recognition of the cancer cells. Once T cells are able to recognize a specific cancer antigen, they will recognize and kill cancer cells that contain that cancer antigen.

[0006] In addition, current technology can only use a limited number of antigens and cannot cover most of the cancer antigens in the patient's body. Due to the diversity of cancer cell antigens and the diversity of cancer mutations, even the same cancer patient will have different cancer cell antigens. Therefore, it is difficult to produce a cancer vaccine that can be used by most people from a limited number of cancer antigens. Cancer cells and tumor tissues from patients each contain unique cancer antigens and mutations, so they belong to personalized antigens, and the cancer vaccines produced also belong to personalized vaccines. However, tumor tissues contain a variety of cancer antigens, and if the whole cell fraction of cancer tissues or cancer cells can be delivered to antigen-presenting cells as a vaccine, the vaccine will have excellent preventive and therapeutic effects against cancer. However, due to the limitations of current technical means, researchers focus on water-soluble components and cannot effectively apply water-insoluble components, which is a difficulty in the current use of whole cell fractions.

[0007] Furthermore, to function as a vaccine, it is necessary that the antigen is first delivered to the antigen-presenting cells, which then process the cancer antigen and present it on the surface of the antigen-presenting cells, and interact with T cells to activate the immune recognition of the T cells against the cancer cells. Once the T cells are able to recognize a specific cancer antigen, they will recognize and kill the cancer cells that contain that cancer antigen. Currently, targeting antigen-presenting cells by particle size is called passive targeting. However, passive targeting alone cannot efficiently present the nanoparticles on the surface of the antigen-presenting cells to interact with T cells. Take a 300 nm nanoparticle as an example, it can be phagocytosed by antigen-presenting cells such as dendritic cells and B cells, and can also be phagocytosed by other cells such as fibroblasts.

[0008] [Contents of the invention] In consideration of this, the object of the present invention is to provide a targeted delivery system carrying a whole cell fraction, which presents the whole cell fraction including soluble and insoluble components of cancer cells or tissues to antigen-presenting cells in an active targeting manner, thereby improving the preventive and therapeutic effects of tumors.

[0009] Another object of the present invention is to provide a use of said targeted delivery system in the manufacture of a vaccine for the prevention and / or treatment of cancer.

[0010] To achieve the above objectives, the present invention provides the following technical solutions:

[0011] A targeted delivery system carrying a whole cell fraction, the whole cell fraction being a nanoscale or microscale particle having a targeting head on its surface, the particle carrying a whole cell fraction of a cancer cell or cancer tissue, the water-insoluble components being dissolved by a solubilizing agent, the whole cell fraction being the water-soluble and water-insoluble components of all cells in the cell or tissue, the targeting head binding to a molecule on the surface of a specific cell or tissue to assist the particle in entering the cell or tissue.

[0012] In the present invention, after dissolving cancer cells or tissues, first obtain water-soluble components that are soluble in pure water or aqueous solution that does not contain a solubilizer, and then dissolve the water-insoluble components in a solubilizing aqueous solution that contains a specific solubilizing agent, so that all cell fractions are converted into components that can be dissolved in aqueous solution, and are carried on the inside and outside of nanoparticles or microparticles to prepare a targeted delivery system, thereby ensuring that most antigenic substances are carried on the prepared targeted delivery system.In practical use, after dissolving cells or tissues, the whole cell fraction can be dissolved directly using a solubilizing aqueous solution that contains a solubilizing agent, without separating the water-soluble components and the water-insoluble components, and the whole cell fraction dissolved in the solubilizing aqueous solution can be used to prepare a targeted delivery system.

[0013] The water-soluble and water-insoluble components of the cell fraction in cancer cells or tumor tissues include the components and fractions of the whole cell. Among them, the non-mutated proteins, polypeptides and genes of the same components as those of normal cells do not cause immune reactions due to the immune tolerance generated in the development process of the autoimmune system, but the mutations of genes, proteins and polypeptides caused by cancer are immunogenic due to the lack of immune tolerance generated in the development process of the autoimmune system, and activate the body's immune response against the cancer cells. These substances with cancer cell-specific immunogenicity produced by disease mutations in the whole cell fraction can be used to prevent and treat cancer.

[0014] In the targeted delivery system according to the present invention, the whole cell fraction can be divided into two parts, a water-soluble component and a water-insoluble component, according to their solubility in pure water or an aqueous solution without a solubilizer. The water-soluble component is the original water-soluble part that is soluble in pure water or an aqueous solution without a solubilizer, and the water-insoluble component is the original water-insoluble part that is insoluble in pure water, and is converted from being insoluble in pure water or an aqueous solution without a solubilizer to being soluble in an aqueous solution containing a solubilizer by using a suitable solubilization method. The water-soluble part and the water-insoluble part of the whole cell fraction can be dissolved by a solubilization aqueous solution containing a solubilizer.

[0015] In the targeted delivery system according to the present invention, the solubilizing agent is urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, an alkaline solution with a pH of more than 7, an acidic solution 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 TMThe solvents include, but are not limited to, dimethylamine oxide, IGEPAL CA-630, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, and ethyl acetate, and one or more of these may be selected.

[0016] The water-insoluble components can also be changed from insoluble to soluble in pure water by other methods that can solubilize proteins and polypeptide fragments. However, in the specific implementation process of the present invention, the effects of urea, guanidine hydrochloride, SDS, sodium deoxycholate or glycerol are obviously superior to other solubilizing agents such as PEG. Preferably, the present invention directly uses a solubilizing solution containing urea or guanidine hydrochloride to directly dissolve cells or tissues and directly dissolve the whole cell fraction. In the embodiment given in the present invention, 8M urea and 6M guanidine hydrochloride aqueous solution are used to dissolve the water-insoluble components of tumor tissues or cancer cells, but in practical use, other solubilizing solutions that can solubilize the water-insoluble components of the whole cell fraction, such as SDS and glycerol, can be used.

[0017] Microparticles or nanoparticles have an optimal particle size for targeting antigen-presenting cells, but the optimal size of nanoparticles or microparticles made from different materials may be different. For example, it is currently believed that the optimal size of cationic liposomes may be about 50-150 nm, and nanoparticles made from PLGA may be about 200-500 nm. And microparticles are theoretically believed to be optimal at 1.5-5 μm. This optimal size varies depending on the material of the particle. Targeting antigen-presenting cells by particle size is called passive targeting.

[0018] In addition to passive targeting, the present invention also uses active targeting strategy, that is, attaching a targeting head molecule that can target specific cells to the outside of nanoparticles or microparticles. This allows nanoparticles or microparticles to be directly targeted to the surface of specific cells or tissues, and helps the particles to enter the cells or tissues by binding with ligand receptors. These cells include, but are not limited to, dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, and basophils in white blood cells, and the tissues that the targeting head can target include, but are not limited to, lymph nodes, thymus, spleen, and bone marrow. Molecules that can target these cells and tissues include, but are not limited to, mannose, mannan, CD32 antibody, CD11c antibody, BDCA-1 antibody, CD141 antibody, CD123 antibody, CD14 antibody, CD19 antibody, CD20 antibody, CD103 antibody, CD11b antibody, CD33 antibody, CD40 antibody, CD40L antibody, CD80 antibody, CD86 antibody.

[0019] For example, when relying only on passive targeting, a 300 nm nanoparticle may be phagocytosed by antigen presenting cells such as dendritic cells, B cells, etc., but may also be phagocytosed by other cells such as fibroblasts. When an active targeting strategy is used, it can be directed to be phagocytosed only by dendritic cells, which are the most important antigen presenting cells. In the enumerated examples of the present invention, nanoparticles or microparticles modified with mannose (i.e., targeting heads) target dendritic cells, and in practical use, any targeting head that can target dendritic cells or other types of specific cells or tissues can be used.

[0020] The particle size of the nano- or micro-vaccine is nano-scale or micro-scale, and the particle size should be within a suitable range to ensure that the vaccine is phagocytosed by antigen-presenting cells and improve the phagocytosis efficiency. In the targeted delivery system described in the present invention, the particle size of the nano-scale size particles is 1 nm to 1000 nm. In some embodiments, the particle size of the nano-sized particles is 30 nm to 800 nm. Furthermore, in some embodiments, the particle size of the nano-sized particles is 50 nm to 600 nm.

[0021] In the targeted delivery system described herein, the microscale-sized particles have a particle size of 1 μm to 1000 μm. In some embodiments, the micro-sized particles have a particle size of 1 μm to 100 μm. In some embodiments, the micro-sized particles have a particle size of 1 μm to 10 μm. Furthermore, in some embodiments, the micro-sized particles have a particle size of 1 μm to 5 μm.

[0022] In the delivery system according to the present invention, the surface of said nano- or micro-sized particles may be electrically neutral, negatively charged or positively charged.

[0023] In order to enhance the immunogenicity and efficacy of the targeted delivery system, immune adjuvants with certain immune modulating functions can also 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, immune enhancing reconstituted influenza virus particles, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, new bovine liver active peptide, miquimod, polysaccharides, curcumin, immune adjuvant poly ICLC, corynebacterium parvum, vaccine), hemolytic streptococcal 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, active ingredients of Chinese herbal medicines such as Ginseng and Astragalus, one or more of which can be selected. The method of adding the immune adjuvant according to the present invention includes carrying it within nanoparticles or microparticles, carrying it on the surface of nanoparticles or microparticles, or carrying it both within nanoparticles or microparticles and on the surface of nanoparticles or microparticles. Preferably, the immune adjuvant is added to the whole cell fraction.

[0024] In a specific embodiment of the present invention, polyinosinic-polycytidylic acid (Poly(I:C)), Bacillus Calmette-Guerin (BCG) or CpG is used as an immune adjuvant and is added to the water-soluble component containing the cancer cell lysate or tumor tissue lysate and the original water-insoluble component soluble in the solubilizing agent, and the concentration of Poly(I:C), BCG or CpG is preferably greater than 1 ng / mL.

[0025] To improve the targeting and efficacy of the targeted delivery system, the present invention also includes adding a PEG overcoat to the outside of the particles.

[0026] In the targeted delivery system according to the present invention, the materials for preparing the nano-scale or micro-scale particles are one or more of organic synthetic polymer materials, natural polymer materials, inorganic materials, bacteria or viruses.

[0027] 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.

[0028] The natural polymeric materials are biocompatible or degradable polymeric materials, including, but not limited to, phospholipids, cholesterol, starch, sugars, polypeptides, sodium alginate, albumin, collagen, gelatin, and cell membrane components.

[0029] The inorganic materials are non-explicitly biotoxic materials, including, but not limited to, ferric oxide, ferric oxide, calcium carbonate, and calcium phosphate.

[0030] The shape of the delivery system according to the present invention may be any common shape, including, but not limited to, a sphere, an ellipse, a barrel, a polygon, a rod, a sheet, a wire, a worm, a square, a triangle, a butterfly, or a disk.

[0031] In the targeted 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 particle, and / or loaded separately or together on the particle surface. It includes, but is not limited to, the water-soluble components are loaded together in the particle and on the particle surface, the water-soluble components are loaded together in the particle and on the particle surface, the water-soluble components are loaded in the particle and the water-insoluble components are loaded on the particle surface, the water-soluble components and the water-insoluble components are loaded on the particle surface, the water-soluble components and the water-insoluble components are loaded on the particle surface, only the water-soluble components are loaded on the particle surface, the water-soluble components are loaded in the particle and the water-soluble components and the water-soluble components are loaded on the particle surface, the water-soluble components are loaded in the particle and the water-soluble components and the water-soluble components are loaded on the particle surface together, the water-soluble components are loaded in the particle and the water-soluble components and the water-soluble components are loaded on the particle surface together, the water-soluble components are loaded in the particle and the water-soluble components and the water-soluble components are loaded on the particle surface together, the water-soluble components and ...

[0032] The schematic diagrams of the structure 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 nanoparticle or microparticle having a specific structure can be used, or multiple nanoparticles or microparticles having different structures can be used simultaneously.

[0033] The targeted delivery system according to the present invention can be prepared according to any previously developed method for preparing nano- and micro-sized particles, including but not limited to the common solvent evaporation method, dialysis method, extrusion method, hot melt method. In some embodiments, the delivery system is prepared by double emulsion method in solvent evaporation method.

[0034] The whole cell fraction targeted delivery system according to 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 through the immunogenicity of the loaded components. Therefore, the present invention also provides the use of said whole cell fraction targeted delivery system in the manufacture of a vaccine for preventing and / or treating cancer.

[0035] Here, the cancer is a solid tumor or a blood system tumor, including, but not limited to, endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, urinary system tumors, immune system tumors, circulatory system tumors, respiratory system tumors, blood system tumors, and skin tumors.

[0036] When used as a cancer vaccine to prevent and treat cancer, the targeted delivery system described in the present invention can be administered multiple times before or after the onset of cancer to activate the body's immune system, thereby slowing the progression of cancer, treating cancer, or preventing the recurrence of cancer.

[0037] As can be seen from the above technical solution, the present invention uses an aqueous solution containing a specific solubilizer to convert the components in cells that are insoluble in pure water or aqueous solutions without solubilizers into soluble components, which can be used to manufacture nanoparticles and microparticles, improving the inclusion and immunogenicity of antigenic substances or components carried by nanoparticles or microparticles. At the same time, a targeting head that can target antigen-presenting cells is added to improve the phagocytosis efficiency of antigen-presenting cells in the manner of active targeting, thereby improving the preventive or therapeutic effect of cancer.

[0038] [Drawing description] FIG. 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 producing a nanovaccine or a microvaccine 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 producing a nanovaccine or a microvaccine.

[0039] 2 to 12 are schematic diagrams of the structure of nano- or micro-sized particles carrying water-soluble and water-insoluble cellular components, in which 1: water-soluble components in the cell or tissue fraction, 2: water-insoluble components in the cell or tissue fraction, 3: immune adjuvant, 4: nanoparticles or microparticles, 5: inner core part in the nanoparticle, and 6: target head that can target a specific cell or tissue. In Figs. 2 to 3, the immune adjuvant is contained on the surface and inside of the nanoparticle or microparticle. In Figs. 4 to 5, the immune adjuvant is distributed only inside the nanoparticle or microparticle. In Figs. 6 to 7, the nanoparticle or microparticle contains the immune adjuvant only on the outer surface. In Figs. 8 to 9, the immune adjuvant is not contained either inside or on the outer surface of the nanoparticle or microparticle. In Fig. 10, the cell fraction and / or the immune adjuvant is distributed only inside the nanoparticle or microparticle. In Fig. 11, the cell fraction and / or the immune adjuvant is distributed only outside the nanoparticle or microparticle. In FIG. 12, the cellular fraction and the immune adjuvant are distributed inside or outside the nanoparticles or microparticles, respectively.

[0040] In Figures 2 to 9, in 2.a to 2.i in Figure 2, 6.a to 6.i in Figure 4, 10.a to 10.i in Figure 6, and 14.a to 14.i in Figure 8, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticle or microparticle are distributed inside the nanoparticle or microparticle, a clear inner core is not formed. In 3.a to 3.i in Figure 2, 7.a to 7.i in Figure 4, 11.a to 11.i in Figure 6, and 15.a to 15.i in Figure 8, the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticle or microparticle are distributed in one inner core part inside the nanoparticle or microparticle. In 4.a to 4.i of Figure 3, 8.a to 8.i of Figure 5, 12.a to 12.i of Figure 7, and 16.a to 16.i of Figure 9, the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticle or microparticle are distributed in multiple inner core portions inside the nanoparticle or microparticle. In 5.a to 5.i of Figure 3, 9.a to 9.i of Figure 5, 13.a to 13.i of Figure 7, and 17.a to 17.i of Figure 9, the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticle or microparticle are distributed in the outer layer of the inner core formed inside the nanoparticle or microparticle.a: the water-soluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles, b: the water-insoluble components of the cell or tissue fraction are carried inside and on the surface of the nanoparticles or microparticles, c: the water-insoluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and both of the water-soluble components of the cell or tissue fraction are carried on the surface, d: the water-soluble components of the cell or tissue fraction are carried inside the nanoparticles or microparticles, and both of the water-insoluble components of the cell or tissue fraction are carried on the surface, e: the water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticles or microparticles, and both of the water-soluble and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticles or microparticles, and f: a: the 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; g: the water-soluble and water-insoluble components of the cell or tissue fraction are carried together inside the nanoparticle or microparticle, and only the water-insoluble 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 together inside the nanoparticle or microparticle, and only the water-soluble and water-insoluble components of the cell or tissue fraction are carried together 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 and water-insoluble components of the cell or tissue fraction are carried together on the surface of the nanoparticle or microparticle.

[0041] In Figures 10 to 12, when the water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles in a, b, and c are distributed inside the nanoparticles or microparticles, they do not form a clear inner core. The water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles in d, e, and f are distributed in one inner core part inside the nanoparticles or microparticles. The water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles in g, h, and i are distributed in multiple inner core parts inside the nanoparticles or microparticles. The water-soluble or water-insoluble components in the cell or tissue fraction carried by the nanoparticles or microparticles in j, k, and l are distributed in the outer layer of the inner core formed inside the nanoparticles or microparticles. The components carried by the nanoparticles or microparticles in a, d, g, and j are all water-soluble components in the cell or tissue fraction. The components carried by the nanoparticles or microparticles in b, e, h, and k are all water-insoluble components in the cell or tissue fraction. The nanoparticles or microparticles in c, f, i and l together carry the water-soluble and water-insoluble components in the cell or tissue fraction.

[0042] 13 to 19 are results of the use in the treatment and prevention of cancer in the case where the whole cell fraction of tumor tissue or cancer cells is supported by the targeted nanoparticles or microparticles in Examples 1 to 7. a, Structure of targeted nanoparticles or microparticles supporting water-soluble components / or water-insoluble components in the whole cell fraction of tumor tissue or cancer cells. b, Structure of targeted nanoparticles or microparticles supporting water-insoluble components in the whole cell fraction of tumor tissue or cancer cells. c, Experimental results of targeted nanovaccines or microvaccines for tumor growth inhibition. d, Results of mouse survival phase experiments. In the experimental results, each data point in the graph of the tumor growth inhibition experiment is the mean ± standard error (mean ± SEM). n=10 for the tumor growth inhibition experiment and the mouse survival phase experiment. The significant difference in the tumor growth inhibition experiment in Figure c is analyzed by ANOVA method. The significant difference in Figure d is analyzed by Kaplan-Meier and log-rank test. * indicates that the group is significantly different from the PBS control group at p<0.05. ☆ indicates that the group is significantly different from the blank nanoparticle or blank microparticle control groups at p<0.05.

number

[0043] [Specific implementation method] The embodiments of the present invention disclose a targeted delivery system carrying a whole cell fraction and its use. Those skilled in the art can refer to the contents of this specification and appropriately improve the process parameters to realize it. It should be specifically pointed out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The targeted delivery system and its use described in the present invention have been described through preferred embodiments, but it is clear that those involved can make modifications or suitable modifications and combinations to the targeted delivery system and its use described in this specification to realize and use the present invention without departing from the content, spirit and scope of the present invention.

[0044] The whole cell fraction delivery system described in the present invention can be used to produce a vaccine for preventing and / or treating cancer, and its production process and application field are as shown in Figure 1. In the production, after dissolving cells or tissues, the water-soluble components and the water-insoluble components can be collected first to produce nano-vaccines or micro-vaccines, respectively; alternatively, a solubilizing solution containing a solubilizing agent can be directly used to directly dissolve cells or tissues, and then the whole cell fraction can be dissolved to produce nano-vaccines or micro-vaccines.

[0045] The method for producing the targeted delivery system (also called nanovaccine or microvaccine) according to the present invention is a general method for production. In some embodiments, the nanovaccine or microvaccine The production of nanoparticles is carried out by using the double emulsion method in the solvent evaporation method. or microparticles The manufacturing materials are organic polymer polylactic acid-glycolic acid copolymer (PLGA) and polylactic acid (PLA). The immunoadjuvants used are poly(I:C), Bacillus Calmette-Guerin vaccine (BCG) or CpG. In the actual use process, those skilled in the art can follow the specific conditions to determine the manufacturing method, manufacturing process, nanoparticles used. or microparticles It is understood that the manufacturing materials, the type and concentration of the immunoadjuvant, etc. can be appropriately adjusted.

[0046] In some embodiments, the specific preparation method of the double emulsion method adopted in the present invention is as follows.

[0047] 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 polymeric material at a second predetermined concentration.

[0048] In some embodiments, the aqueous phase solution contains each fraction in the cancer cell lysate and an immune enhancing adjuvant such as poly(I:C), BCG, or CpG, and each fraction in the cancer cell lysate is prepared by adding a water-soluble component or 8 M urea to the aqueous phase solution. or other solubilizing agents The concentration of the water-soluble component derived from the cancer cells contained in the aqueous phase solution or the concentration of the water-insoluble component derived from the cancer cells dissolved in 8M urea, i.e., the first predetermined concentration, must have a protein polypeptide concentration content of more than 1ng / mL. The reason for selecting such a concentration is that, in general, the higher the concentration of the aqueous solution of the cell lysate used, the higher the concentration of the nanoparticles produced. or microparticles This is because the inventors have found through numerous experiments that when the concentration of the produced protein polypeptide exceeds 1 ng / mL, i.e., the first predetermined concentration exceeds 1 ng / mL, sufficient cancer antigens can be carried and the associated immune response can be activated. The concentration of the immune adjuvant in the initial aqueous phase is higher than 0.01 ng / mL. Also, the first predetermined volume is 600 μL.

[0049] In some embodiments, the aqueous phase solution contains each fraction in the tumor tissue lysate and an immune enhancing adjuvant such as poly(I:C), BCG or CpG, and each fraction in the tumor tissue lysate is prepared by adding a water-soluble component or 8 M urea to the aqueous phase solution. or other solubilizing agentsThe concentration of the water-soluble components derived from the tumor tissue contained in the aqueous phase solution or the concentration of the water-insoluble components derived from the tumor tissue dissolved in 8M urea, i.e., the first predetermined concentration, must be greater than 1ng / mL in protein polypeptide concentration. The reason for selecting such a concentration is that, in general, the higher the concentration of the aqueous solution of tumor tissue lysate used, the higher the concentration of the nanoparticles produced. or microparticles This is because the inventors have found through numerous experiments that when the concentration of the produced protein polypeptide exceeds 1 ng / mL, i.e., the first predetermined concentration exceeds 1 ng / mL, sufficient cancer antigens can be carried and the associated immune response can be activated. The concentration of the immune adjuvant in the initial aqueous phase is higher than 0.01 ng / mL. Also, the first predetermined volume is 600 μL.

[0050] In the present invention, the medical polymer material is dissolved in an organic solvent to obtain an organic phase containing the medical polymer material in a second predetermined volume and a second predetermined concentration. In some embodiments, the medical polymer material is PLGA modified into a target head, a mixture of PLGA and PLA, the organic solvent is selected as dichloromethane, and the volume of the obtained organic phase, i.e., the second predetermined volume, is 2 mL. Furthermore, in some embodiments, the range of the second predetermined concentration of the medical polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0051] In the present invention, the reason for selecting PLGA and PLGA modified with targeting head is that the material is a biodegradable material and has been approved by the FDA for use as a drug dressing; in practical use, a mixture of other materials that can be used to prepare nano-sized particles or micro-sized particles and the targeting head modified material can also be selected.

[0052] In practice, the second predetermined volume of the organic phase is set according to the 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 first predetermined volume, the second predetermined volume, and the ratio between the first predetermined volume and the second predetermined volume can be adjusted as necessary to produce nanoparticles. or microparticles The size can be adjusted.

[0053] 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.

[0054] The purpose of this step is to Or micronization The length of ultrasonic time or the stirring speed and time can control the size of the nanoparticles produced, and 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.

[0055] Step 3: The mixture obtained after the treatment of 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.

[0056] 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 the mixture. Or micronization To make.

[0057] 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 the ratio to the second predetermined volume. In the present invention, the ratio range of the second predetermined volume to the third predetermined volume is set to 1:1.1 to 1:1000, and may be preferably 2:5. In a specific implementation process, the ratio of the second predetermined volume to the third predetermined volume can be adjusted to control the size of the nanoparticles.

[0058] Similarly, the ultrasonic time or stirring time in this step, and the volume and concentration of the emulsifier aqueous solution are all adjusted to produce nanoparticles of an appropriate size. or microparticles Take the value to get.

[0059] 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 a predetermined stirring condition is met.

[0060] In this step, the aqueous emulsifier solution is still PVA and the fourth predetermined volume is greater than 50 mL.

[0061] The fourth predetermined concentration is 5 mg / mL, and the fourth predetermined concentration is a concentration of nanoparticles of an appropriate size. or microparticles The selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the fourth predetermined volume to the third predetermined volume is in the range of 1:1.5 to 1:2000, preferably 1:10. In a specific implementation process, the nanoparticles or microparticles The ratio of the third and fourth predetermined volumes can be adjusted to control the size of the third and fourth predetermined volumes.

[0062] 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.

[0063] Step 5: The mixture that satisfies the specified stirring conditions and is processed in step 4 is centrifuged at a rotation speed exceeding 100 RPM for 1 minute or more, 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 freeze-drying protection agent or a sixth predetermined volume of PBS (or physiological saline).

[0064] 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 microparticlesExperiments relating to the adsorption of cancer cell lysates onto the surface can be directly performed.

[0065] In some embodiments of the present invention, the precipitate obtained in step 5 needs to be lyophilized when it is resuspended in an aqueous solution containing a lyoprotectant, and after lyophilization, the nanoparticles or microparticles To conduct experiments related to the adsorption of cancer cell lysate onto the surface of

[0066] In the present invention, the freeze-drying protection agent is selected to be trehalose.

[0067] In the present invention, the fifth predetermined volume of the lyophilization protection agent in this step is 20 mL, and the fifth predetermined concentration is 4 mass %, in order not to affect the lyophilization effect during the subsequent lyophilization.

[0068] Step 6: The suspension containing the lyoprotectant obtained in step 5 is freeze-dried and the lyophilisate is stored for use.

[0069] Step 7: Resuspend the nanoparticles obtained in step 5 in a sixth predetermined volume of PBS (or saline). (or microparticles) or the nanoparticles after lyophilization obtained in step 6 using a sixth predetermined volume of PBS (or physiological saline). (or microparticles) The lyophilized material containing the lyophilization protection agent is resuspended and mixed with a seventh predetermined volume of the water-soluble component or the original water-insoluble component dissolved in 8 M urea, and then allowed to stand for 0.1 minutes or more to obtain a nanovaccine or microvaccine.

[0070] In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, a preferable volume ratio is 1:100 to 100:1, and an optimum volume ratio is 1:30 to 30:1.

[0071] In some embodiments, the resuspended 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.

[0072] In some embodiments, as shown in FIG. 2, the present invention first solubilizes the water-soluble or (and) water-insoluble portion of the cell fraction, which is soluble in pure water, with a solubilizing agent, and then loads the water-soluble or (and) water-insoluble portion of the cell fraction into nanoparticles or microparticles, together with the immune enhancing agent; and then loads the water-soluble or (and) water-insoluble portion of the cell fraction into nanoparticles or microparticles. or microparticles The surface of the nanoparticle or microparticle is adsorbed with the immune enhancing agent. In this way, the loading capacity of the water-soluble or water-insoluble components of the cells in the nanoparticle or microparticle can be maximized. In the actual use process, the solubilizing solution containing the solubilizing agent (8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution) can also be directly used to directly dissolve the cells or tissues, and the whole cell fraction can be directly dissolved before producing the nanovaccine or microvaccine.

[0073] The following further describes the whole cell fraction loaded targeted delivery system provided by the present invention and its uses.

[0074] Example 1. Whole cell fraction of melanoma tumor tissue loaded inside mannose-modified nanoparticles for use in cancer therapy In this example, a nano vaccine carrying a whole cell fraction of tumor tissue was prepared using mouse melanoma as a cancer model, and a method for treating melanoma using the vaccine was described. In actual use, the specific dosage form, adjuvant, administration time, administration frequency, and administration method can be adjusted according to the situation.

[0075] In this example, B16-F10 mouse melanoma was used as a cancer cell model. In this example, a nanovaccine was produced by loading the soluble components of mouse melanoma tumor tissue inside and on the surface of nanoparticles. First, a mouse melanoma tumor mass was obtained and dissolved to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, a nanovaccine was produced by loading the water-soluble and water-insoluble components of the tumor tissue lysate using PLGA (50:50) and PLA as nanoparticle scaffolding materials and CpG as an immune adjuvant, employing a solvent volatilization method. The nanovaccine was then used to treat tumors in melanoma-bearing mice.

[0076] (1) Lysis of tumor tissue and collection of each fraction Each C57BL / 6 mouse was subcutaneously inoculated with 150,000 B16F10 melanoma cells on the back, and the inoculated tumors of each mouse were 400 mm 3 From 1500mm 3 When the tumor tissues had grown to a volume of 1000 mg / kg, the mice were killed to collect the tumor tissues. The tumor tissues were cut and polished, and an appropriate amount of pure water was added through a cell strainer, followed by freezing, thawing, and sonication at least five times. After the cells in the tumor tissue site were lysed, the cell lysate of the tumor mass tissue was centrifuged at a rotation speed of over 12,000 RPM for 5 minutes to obtain the supernatant, i.e., the water-soluble components in the tumor mass tissue that are soluble in pure water; the resulting precipitate was dissolved by adding an 8M urea aqueous solution, which allowed the original water-insoluble components insoluble in pure water to be converted into soluble components in 8M urea aqueous solution. The water-soluble components of the tumor tissue lysate obtained and the original water-insoluble components dissolved in 8M urea were the raw material source for producing nanovaccines for treating cancer cells.

[0077] (2) Manufacturing nanovaccines In this example, the nanovaccine and blank nanoparticles as a control were prepared by using a double emulsion method in a solvent evaporation method, the molecular weight of the nanoparticle preparation material PLGA (50:50) used was 24KDa-38KDa, the molecular weight of the mannose-modified PLGA (50:50) used was 24KDa-38KDa, and the molecular weight of the PLA used was 10KDa. The mass ratio of unmodified PLGA, mannose-modified PLGA, and PLA was 8:1:2. The immune adjuvant used was CpG, and CpG was distributed inside the nanoparticles. The preparation method was as described above. The average particle size of the nanoparticles was about 280nm, and the average surface potential Zeta potential of the nanoparticles was about -8mV. 60μg of protein or polypeptide fraction was carried per 1mg of PLGA nanoparticles, and the CpG immune adjuvant used inside and outside per 1mg of PLGA nanoparticles was 0.01mg, half inside and half outside. The particle size of the blank nanoparticles was about 240 nm, and in the preparation of the blank nanoparticles, pure water or 8M urea containing the same amount of CpG was used to replace the corresponding water-soluble and water-insoluble components, respectively.

[0078] (3) Using nanovaccines for cancer treatment The experimental groups used in this study were set up as follows: nanovaccine group, PBS control group, blank nanoparticle + organization Lysate group.

[0079] Six to eight week old female C57BL / 6 mice were selected as model mice to prepare melanoma-bearing mice.

[0080] The administration method of the nano vaccine group was as follows: on day 0, 150,000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 200μL of 2mg PLGA nanoparticles carrying the water-soluble components of the cancer cell lysate on the inside and surface and 200μL of 2mg PLGA nanoparticles carrying the original water-insoluble components dissolved in 8M urea on the inside and surface were subcutaneously injected, respectively.

[0081] The procedure for the PBS blank control group was as follows: on day 0, 150,000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 400 μL of PBS was subcutaneously injected, respectively.

[0082] Blank nanoparticles + tissue lysate control group: 150000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse on day 0, and the same amount of water-soluble components in the tumor tissue lysate, the same amount of the original water-insoluble components dissolved in 8M urea in the lysate, and 4mg of PLGA blank nanoparticles loaded with the same amount of CpG but without lysate components were subcutaneously injected on days 4, 7, 10, 15, and 20, respectively. To avoid free lysate adsorption on the surface of the blank nanoparticles, the above three should be administered separately and injected at different sites.

[0083] 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, mice were not allowed to grow beyond a tumor volume of 2000 mm in the in-life study. 3 If the body weight exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0084] (4) Experimental results As shown in FIG. 13, the PBS blank control group and the blank nanoparticle + organization Compared with the lysate control group, the tumor volume growth rate of mice in the nanovaccine group was significantly slower (p<0.05), and the survival time of mice was significantly extended (p<0.05). This indicates that the nanovaccine carrying the water-soluble and water-insoluble components of cancer cells described in the present invention has a therapeutic effect against melanoma and can partially cure melanoma.

[0085] Example 2. Whole cell fraction of melanoma tumor tissue is loaded into and onto mannose-modified microparticles for use in cancer therapy In this embodiment, the B16-F10 mouse melanoma is used as a cancer model to describe the method of treating melanoma using microvaccines. In practical use, the specific dosage form can be adjusted according to circumstances.

[0086] In this example, all the dissolved components of mouse melanoma tumor tissue were loaded together inside and on the surface of microparticles to produce a microvaccine. First, mouse melanoma tumor mass was obtained and dissolved to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, PLGA (50:50) was added to the microparticles. Microparticles Using poly(I:C) as the scaffold material and poly(I:C) as the immune adjuvant, the solvent evaporation method was used to prepare a microvaccine that co-loaded the soluble and insoluble components of tumor tissue lysate, and the microvaccine was then used to treat tumors in melanoma-bearing mice.

[0087] (1) Lysis of tumor tissue and collection of each fraction The processing method is the same as in the first embodiment.

[0088] (2) Production of microvaccines In this example, the microvaccines and blank microparticles as controls were prepared by the double emulsion method in the solvent evaporation method, the molecular weight of the microparticle preparation material PLGA used was 24KDa-38KDa, and the molecular weight of the mannose-modified PLGA (50:50) used was 24KDa-38KDa. The mass ratio of unmodified PLGA and mannose-modified PLGA (50:50) was 10:1. The immune adjuvant used was poly(I:C), and poly(I:C) was distributed inside the microparticles. The preparation method was as described above, and the water-soluble components and the water-insoluble components were supported together on the same microparticles. In this example, the whole cell fraction was supported together on the inside and the surface of the microparticles. When the whole cell fraction is supported on the surface of the microparticles, the microparticles supported with the whole cell fraction and the cell components may be mixed in a volume ratio of 10:1 and then allowed to stand for 15 minutes. The average particle size of the microparticles was about 2.0 μm, Microparticles The average surface potential Zeta potential of the PLGA microparticles was approximately -17 mV. 70 μg of protein or polypeptide fraction was loaded per 1 mg of PLGA microparticles, and the amount of poly(I:C) immune adjuvant used on the inside and outside per 1 mg of PLGA microparticles was 0.01 mg, half each on the inside and outside. The particle size of the blank microparticles was approximately 1.8 μm, and in the preparation of the blank microparticles, the same amount of poly(I:C) in pure water or 8 M urea was used to replace the corresponding water-soluble and water-insoluble components, respectively.

[0089] (3) Using microvaccines for cancer treatment The experimental groups in this study were set up as follows: microvaccine group, PBS control group, blank microparticles + cell lysate group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice.

[0090] The administration method of the microvaccines was as follows: on day 0, 150,000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 400 μL of 4 mg PLGA microvaccines, which were loaded with both the water-soluble and water-insoluble components of the cancer cell lysate on the inside and surface, were subcutaneously injected.

[0091] The protocol for the PBS blank control group was as follows: on day 0, 150,000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse, and on days 4, 7, 10, 15, and 20, 400 μL of PBS was subcutaneously injected.

[0092] Blank microparticles + cell lysate control group: 150,000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse on day 0, and 4 mg of PLGA blank nanoparticles loaded with the same amount of soluble + insoluble components of cancer cell lysate and the same amount of poly(I:C) but without cell lysate components were subcutaneously injected on days 4, 7, 10, 15, and 20. To avoid free cell lysate adsorption on the surface of blank microparticles, the above two should be administered separately and injected at different sites.

[0093] 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.

[0094] (4) Experimental results As shown in Figure 14, compared with the PBS blank control group and the blank microparticle + cell lysate control group, the tumor volume growth rate of mice in the microvaccine group was significantly slower (p<0.05), and the survival time of mice was significantly extended (p<0.05). This shows that the microvaccine 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.

[0095] Example 3. Whole cell fractions of melanoma tumor tissue are loaded into and onto mannose-modified nanoparticles for use in cancer prevention In this example, a mouse melanoma was used as a cancer model, and a nanovaccine carrying a whole cell fraction of tumor tissue was produced and used to treat melanoma. prevention In practical use, the specific dosage form, adjuvant, administration time, administration frequency, and administration method can be adjusted according to the situation.

[0096] In this example, B16-F10 mouse melanoma was used as a cancer cell model. In this example, a nanovaccine was produced by loading soluble components of mouse melanoma tumor tissue inside and on the surface of nanoparticles. First, a mouse melanoma tumor mass was obtained and dissolved to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, a nanovaccine was produced by loading the water-soluble and water-insoluble components of the tumor tissue lysate using PLGA (50:50) as the nanoparticle backbone material and poly(I:C) as the immune adjuvant, employing a solvent evaporation method. The nanovaccine was then used to immunize tumors in melanoma-bearing mice. prevention did.

[0097] (1) Lysis of tumor tissue and collection of each fraction The method is the same as in Example 1.

[0098] (2) Manufacturing nanovaccines In this example, the nanovaccine and blank nanoparticles as a control were manufactured by using a double emulsion method in a solvent evaporation method, the molecular weight of the nanoparticle manufacturing material PLGA used was 24KDa-38KDa, and the molecular weight of the mannose-modified PLGA used was 24KDa-38KDa. The mass ratio of unmodified PLGA to mannose-modified PLGA was 10:1. The immune adjuvant used was poly(I:C), and poly(I:C) was distributed inside the nanoparticles. In this example, the manufacturing method is as described above, except that the whole cell fraction is supported on the inside and surface of the nanoparticles together. When the whole cell fraction is supported on the surface of the nanoparticles, the nanoparticles supported with the whole cell fraction and the cell components may be mixed in a volume ratio of 10:1 and then allowed to stand for 15 minutes. The average particle size of the nanoparticles was about 300 nm, and the average surface potential Zeta potential of the nanoparticles was about -8mV. Each mg of PLGA nanoparticles was loaded with 60 μg of protein or polypeptide fraction, and the amount of poly(I:C) immune adjuvant used inside and outside each mg of PLGA nanoparticles was 0.01 mg, half inside and half outside. The particle size of the blank nanoparticles was about 260 nm, and in the preparation of 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.

[0099] (3) Using nanovaccines to prevent cancer Melanoma-bearing mice were prepared by selecting 6-8 week-old female C57BL / 6 mice. On the 42nd, 35th, 28th, 21st, and 14th days before inoculation with B16F10 cancer cells, 200μL of 2mg of PLGA nano vaccine with water-soluble components in the cancer cell lysate on the inside and surface and 200μL of 2mg of PLGA nano vaccine with original water-insoluble components dissolved in 8M urea on the inside and surface were subcutaneously injected. 14 days after the final injection of the nano vaccine, 150,000 B16-F10 cells were subcutaneously inoculated into the lower right back of each mouse, and the day was set as day 0 of tumor cell inoculation. In this experiment, the procedure for the PBS blank control group was as follows. On the 42nd, 35th, 28th, 21st, and 14th days before inoculation with B16-F10 cancer cells, 400μL of PBS was subcutaneously injected. On day 0, each mouse was inoculated subcutaneously with 150,000 B16-F10 cells in the lower right back.

[0100] 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.

[0101] (4) Experimental results As shown in Figure 15, the tumor growth rate in the nanovaccine vaccination group was significantly slower (p<0.05) and the survival time of the mice was significantly extended (p<0.05) compared with the PBS control group. This indicates that the nanovaccine carrying the water-soluble and water-insoluble components of cancer cells with dendritic cell targeting ability described in the present invention has a preventive effect against melanoma.

[0102] Example 4: Whole cell fraction of lung cancer tumor tissue is loaded inside mannose-modified nanoparticles and used for lung cancer prevention In this example, a nano vaccine carrying a whole cell fraction of tumor tissue was prepared using a mouse lung cancer as a cancer model, and a method for preventing lung cancer using the vaccine was described. In actual use, the specific dosage form, adjuvant, administration time, administration frequency, and administration method can be adjusted according to the situation.

[0103] In this example, LLC mouse lung cancer was used as a cancer cell model. In this example, a nanovaccine was produced by loading soluble components of mouse lung cancer tumor tissue inside and on the surface of nanoparticles. First, a mouse lung cancer tumor mass was obtained and dissolved to produce the water-soluble components of the tumor mass tissue and the original water-insoluble components dissolved in 8M urea. Next, a nanovaccine was produced by loading the water-soluble and water-insoluble components of the tumor tissue lysate using PLGA (50:50) as the nanoparticle backbone material and poly(I:C) as the immune adjuvant, employing a solvent evaporation method. The nanovaccine was then used to suppress tumor growth in lung cancer-bearing mice. prevention did.

[0104] (1) Lysis of tumor tissue and collection of each fraction 1 million cells in the flank of each C57BL / 6 mouse LLC cells The tumors in each mouse were subcutaneously inoculated with 3 From 1500mm 3 When the tumor tissue had grown to a volume of 1000 mg / kg, the mice were killed and the tumor tissue was collected. The tumor tissue was cut and polished, and an appropriate amount of pure water was added through a cell strainer, followed by freezing, thawing, and sonication at least five times. After the cells in the tumor tissue site were lysed, the cell lysate of the tumor mass tissue was centrifuged at a rotation speed of over 12,000 RPM for 5 minutes to obtain the supernatant, i.e., the water-soluble components in the tumor mass tissue that are soluble in pure water. The resulting precipitate was dissolved by adding an 8M urea aqueous solution, which converted the original water-insoluble components that were insoluble in pure water into those that were soluble in the 8M urea aqueous solution. The water-soluble components of the tumor tissue lysate obtained and the original water-insoluble components dissolved in 8M urea were used to convert the cancer cells into the water-soluble components that were insoluble in the 8M urea aqueous solution. prevention The study was a source of raw materials for producing nanovaccines to treat the disease.

[0105] (2) Manufacturing nanovaccines In this example, the nanovaccine and blank nanoparticles as a control were prepared by using a double emulsion method in a solvent evaporation method, the molecular weight of the nanoparticle preparation material PLGA used was 24KDa-38KDa, and the molecular weight of the mannose-modified PLGA used was 24KDa-38KDa. The mass ratio of unmodified PLGA and mannose-modified PLGA was 10:1. The immune adjuvant used was poly(I:C), and poly(I:C) was distributed inside the nanoparticles. The preparation method was as described above. The average particle size of the nanoparticles was about 300nm, and the average surface potential Zeta potential of the nanoparticles was about -8mV. 60μg of protein or polypeptide fraction was carried per 1mg of PLGA nanoparticles, and the poly(I:C) immune adjuvant used on the inside and outside per 1mg of PLGA nanoparticles was 0.01mg, half inside and half outside. The particle size of the blank nanoparticles was approximately 260 nm, and in the preparation of 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.

[0106] (3) Using nanovaccines to prevent lung cancer Female C57BL / 6 mice aged 6 to 8 weeks were selected as model mice to prepare lung cancer-bearing mice.

[0107] The administration method for the nanovaccine group was as follows. On days -49, -42, -35, -28, and -14 before tumor cell inoculation, 200μL of 2mg PLGA nanovaccine with water-soluble components in the cancer cell lysate loaded on its inside and on its surface, and 200μL of 2mg PLGA nanovaccine with the original water-insoluble components dissolved in 8M urea loaded on its inside and on its surface were subcutaneously injected. The PBS control group was administered 2mg PLGA nanovaccine with water-soluble components in the cancer cell lysate loaded on its inside and on its surface on the corresponding days. 400μL On day 0, each mouse was subcutaneously inoculated with 1,000,000 LLC lung cancer cells in the lower right back.

[0108] 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 * b2 where v is the tumor volume, a is the tumor length, and b is the tumor width.

[0109] (4) Experimental results As shown in Figure 16, compared with the PBS blank control group, the tumor growth rate of mice in the nanovaccine group was significantly slower (p<0.05), and the survival time of mice was significantly extended (p<0.05). This indicates that the nanovaccine carrying the water-soluble and water-insoluble components of cancer cells described in the present invention has a preventive effect against lung cancer.

[0110] Example 5. Whole cell fractions of breast cancer cells are loaded inside mannose-modified nanoparticles for use in cancer therapy In this example, a method for preparing a nanovaccine carrying a whole cell fraction and using the vaccine to treat breast cancer in mice was described. In this example, 4T1 mouse triple negative breast cancer cells were used as a cancer cell model. First, 4T1 cells were dissolved to prepare the water-soluble and water-insoluble components of 4T1 cells. Next, PLGA and mannose-modified PLGA (50:50) were used as nanoparticle scaffold materials, and a solvent evaporation method was used to prepare a nanovaccine carrying the water-soluble and water-insoluble components of 4T1 cells. Bacillus Calmette-Guerin (BCG) adjuvant was used as an immune adjuvant, and the nanovaccine was used to treat tumors in 4T1 breast cancer-bearing mice.

[0111] (1) Lysis of cancer cells and collection of each fraction A fixed amount of 4T1 cells was collected, the medium was removed, and the cells were frozen at -20°C to -273°C, and a predetermined amount of ultrapure water was added, followed by repeated freezing and thawing three or more times, optionally accompanied by ultrasonic waves to break the lysed cells. After cell lysis, the lysate was centrifuged at a rotation speed of 100g or more for one minute or more, and the supernatant, i.e., the water-soluble components in 4T1 that are soluble in pure water, were taken, and 8M urea was added to the resulting precipitate to dissolve the precipitate, and the water-insoluble components in 4T1 that are insoluble in pure water were solubilized in an 8M urea aqueous solution. The water-soluble components derived from the cancer cell lysate obtained and the original water-insoluble components dissolved in 8M urea were the raw material source for producing a nano vaccine for cancer treatment.

[0112] (2) Manufacturing nanovaccines The manufacturing method of the nanovaccine in this embodiment and the materials used are basically the same as those in Example 4, except that LLC cells were replaced with 4T1 cells and the adjuvant was replaced with BCG adjuvant.

[0113] (3) Using nanovaccines for cancer treatment Male BALB / c mice aged 6 to 8 weeks were selected to generate 4T1 tumor-bearing mice.

[0114] The administration method of the nano vaccine group 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, 200μL of 2mg PLGA nanoparticles with water-soluble components of cancer cell lysate on the inside and surface + 0.5mg BCG adjuvant, and 200μL of 2mg PLGA nanoparticles with water-insoluble components dissolved in 8M urea on the inside and surface + 0.5mg BCG adjuvant were subcutaneously injected, respectively.

[0115] The protocol for 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 on days 4, 7, 10, 15, and 20, 400 μL of PBS was subcutaneously injected, respectively.

[0116] Blank nanoparticles + cell lysate control group: 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse on day 0, and the water-soluble components in the cancer cell lysate, the original water-insoluble components dissolved in 8M urea in the cancer cell lysate, the same amount of BCG, and 4mg of PLGA blank nanoparticles were subcutaneously injected on days 4, 7, 10, 15, and 20, respectively. In order to avoid the adsorption of free cell lysate on the surface of the blank nanoparticles, the above three should be administered separately and injected at different sites.

[0117] 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 length of the tumor, and b is the width of the tumor. In mouse survival studies, mice with tumor volumes of 2000 mm 3 If the body weight exceeded 100 mg / kg, the mouse was considered dead and was euthanized.

[0118] (4) Experimental results As shown in Figure 17, the tumor growth rate in the nanovaccine administration group was significantly slower (p<0.05) and the survival time of the mice was significantly extended (p<0.05) compared with the PBS blank control group and the blank nanoparticles + cell lysate control group, 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.

[0119] Example 6. Whole cell fractions of breast cancer cells are loaded inside mannose-modified nanoparticles for use in cancer therapy In this example, a method for treating breast cancer in mice was described in which a nanovaccine carrying a whole cell fraction was prepared and the vaccine was used to treat breast cancer. In this example, 4T1 mouse triple negative breast cancer cells were used as a cancer cell model. First, 4T1 cells were dissolved to prepare the water-soluble and water-insoluble components of 4T1 cells. Next, PLGA and mannose-modified PLGA (50:50) were used as nanoparticle scaffold materials, and a solvent evaporation method was used to prepare a nanovaccine carrying the water-soluble and water-insoluble components of 4T1 cells. Bacillus Calmette-Guerin (BCG) adjuvant was used as an immune adjuvant, and the nanovaccine was used to treat tumors in 4T1 breast cancer-bearing mice.

[0120] (1) Lysis of cancer cells and collection of each fraction The manufacturing method is the same as in Example 5.

[0121] (2) Manufacturing nanovaccines The manufacturing method of the nanovaccine in this example and the materials used were basically the same as those in Example 5, and the whole cell fraction was supported only inside the nanoparticles. In the vaccine group containing a PEG protective film, 3.5% PEG-PLGA (the molecular weight of PEG is 5000) was added to the PLGA organic phase during the manufacturing process. PLGA (The molecular weight of this part is 25,000) was added.

[0122] (3) Using nanovaccines for cancer treatment Male BALB / c mice aged 6 to 8 weeks were selected to generate 4T1 tumor-bearing mice.

[0123] The administration methods of the nano vaccine group containing PEG protective film and the nano vaccine group not containing PEG protective film were 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, 200μL of 2mg PLGA nanoparticles carrying water-soluble components + 0.5mg BCG adjuvant and 200μL of 2mg PLGA nanoparticles carrying original water-insoluble components + 0.5mg BCG adjuvant were subcutaneously injected, respectively.

[0124] The protocol for 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 on days 4, 7, 10, 15, and 20, 400 μL of PBS was subcutaneously injected, respectively.

[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.

[0126] (4) Experimental results As shown in Figure 18, compared with the PBS blank control group and the nanovaccine group without PEG protective coating, the tumor growth rate in the nanovaccine group with PEG protective coating was significantly slower (p<0.05), and the survival time of mice was significantly extended (p<0.05). This indicates that the PEG protective coating can prevent nanoparticles from being phagocytosed by cells other than dendritic cells, and extend the circulation time of nanoparticles in the body, thereby improving the targeting and efficacy of nanovaccine.

[0127] Example 7: Solubility of different solubilizers in water-insoluble components In this example, a method for treating breast cancer in mice was described in which a nanovaccine carrying a whole cell fraction was prepared and the vaccine was used to treat breast cancer. In this example, 4T1 mouse triple negative breast cancer cells were used as a cancer cell model. First, 4T1 cells were dissolved to prepare the water-soluble and water-insoluble components of 4T1 cells. The water-insoluble components were dissolved using 6M guanidine hydrochloride and PEG (5000KD), respectively. Next, nanovaccine was prepared using PLGA (50:50) and mannose-modified PLGA (50:50) as nanoparticle scaffold materials. Bacillus Calmette-Guerin vaccine (BCG) adjuvant was used as an immune adjuvant, and the nanovaccine was used to treat tumors in 4T1 breast cancer-bearing mice.

[0128] (1) Lysis of cancer cells and collection of each fraction The preparation method is the same as that of Example 5. The difference is that in this example, 6M guanidine hydrochloride and PEG (5000KD) are used to dissolve the water-insoluble components in the lysed cancer cells, respectively. The solubilizing ability of 6M guanidine hydrochloride for water-insoluble components is obviously stronger than that of PEG (5000KD), and the concentration of 6M guanidine hydrochloride that solubilizes water-insoluble components can reach 80mg / mL, while PEG (5000KD) can only solubilize water-insoluble components to 1mg / mL.

[0129] (2) Manufacturing nanovaccines The manufacturing method of the nanovaccine in this embodiment, the materials used, etc. are basically the same as those in Example 5, and the whole cell fraction was only supported inside the nanoparticles. Furthermore, when manufacturing a nanovaccine carrying a water-insoluble component, the concentration of the original water-insoluble component solubilized by 6M guanidine hydrochloride was 60 mg / mL, and the concentration of the original water-insoluble component solubilized by PEG was 1 mg / mL. Therefore, the nanoparticles carrying the water-insoluble component after solubilizing the manufactured nanovaccine with 6M guanidine hydrochloride carried 70 μg of protein / polypeptide component per 1 mg of PLGA nanoparticles, and the nanoparticles carrying the water-insoluble component after solubilizing with PEG carried 2 μg of protein / polypeptide component per 1 mg of PLGA nanoparticles.

[0130] (3) Using nanovaccines for cancer treatment Female BALB / c mice aged 6 to 8 weeks were selected to generate 4T1 tumor-bearing mice.

[0131] The administration method for the nanovaccine group was as follows: on days -49, -42, -35, -28, and -14 before tumor cell inoculation, 200 μL of 2 mg of PLGA nanoparticles (0.5 mg of BCG adjuvant) carrying water-soluble components of the cancer cell lysate on the inside and on the surface of the nanovaccine were administered, and 200 μL of 2 mg of PLGA nanoparticles (0.5 mg of BCG adjuvant) carrying water-soluble components of the cancer cell lysate on the inside and on the surface of the nanovaccine were administered, respectively. 6M guanidine hydrochloride or PEGThe mice were subcutaneously injected with 2 mg of PLGA nanoparticles (0.5 mg of BCG adjuvant) loaded with the original water-insoluble components dissolved in PBS. The PBS control group was injected with 400 μL of PBS on the corresponding day. On day 0, 400,000 4T1 cells were subcutaneously inoculated into the lower right back of each mouse. 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 as in Example 1.

[0132] (4) Experimental results As shown in Figure 19, compared with the PBS blank control group and the nanovaccine group prepared by solubilizing the water-insoluble components with PEG, the tumor growth rate in the nanovaccine group prepared by solubilizing the water-insoluble components with 6M guanidine hydrochloride was significantly slower (p<0.05), and the survival time of the mice was significantly extended (p<0.05). This indicates that the solubilization solution and method are important for improving the efficacy of nanovaccines.

[0133] The above is for understanding the method of the present invention and its core idea, and it is pointed out that those skilled in the art can improve and modify the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the present invention. [Brief description of the drawings]

[0134] [Figure 1] FIG. 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 producing a nanovaccine or a microvaccine 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 producing a nanovaccine or a microvaccine. [Diagram 2] FIG. 2 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Diagram 3] FIG. 3 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 4]FIG. 4 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Diagram 5] FIG. 5 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 6] FIG. 6 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 7] FIG. 7 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 8] FIG. 8 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 9] FIG. 9 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 10] FIG. 10 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 11] FIG. 11 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 12] FIG. 12 is a schematic diagram of the structure of nano- or micro-sized particles loaded with water-soluble and water-insoluble cellular components. [Figure 13] FIG. 13 shows the results of use in the treatment and prevention of cancer in Example 1, when the whole cell fraction of tumor tissue or cancer cells is carried on targeted nanoparticles or microparticles. [Figure 14] FIG. 14 shows the results of use in the treatment and prevention of cancer in Example 2, when the whole cell fraction of tumor tissue or cancer cells is loaded onto targeted nanoparticles or microparticles. [Figure 15] FIG. 15 shows the results of use in the treatment and prevention of cancer in Example 3, when the whole cell fraction of tumor tissue or cancer cells is carried on targeted nanoparticles or microparticles. [Figure 16] FIG. 16 shows the results of use in the treatment and prevention of cancer in Example 4, when the whole cell fraction of tumor tissue or cancer cells is loaded onto targeted nanoparticles or microparticles. [Figure 17] FIG. 17 shows the results of use in the treatment and prevention of cancer in Example 5, when the whole cell fraction of tumor tissue or cancer cells is loaded onto targeted nanoparticles or microparticles. [Figure 18] FIG. 18 shows the results of use in the treatment and prevention of cancer in Example 6, when the whole cell fraction of tumor tissue or cancer cells is loaded onto targeted nanoparticles or microparticles. [Figure 19] FIG. 19 shows the results of use in the treatment and prevention of cancer in Example 7, when the whole cell fraction of tumor tissue or cancer cells is loaded onto targeted nanoparticles or microparticles.

Claims

1. A nanoscale or microscale particle having a targeting head on its surface, the particle carrying a total cell fraction of a cancer cell or cancer tissue, the total cell fraction being the water-soluble and water-insoluble components of all cells in the cell or tissue, the water-insoluble components being dissolved by a solubilizing agent or a solubilizing aqueous solution containing a solubilizing agent, the targeting head binding to a molecule on the surface of a particular cell or tissue to assist the particle in entering the cell or tissue; the solubilizing agent is urea and / or guanidine hydrochloride; A targeted delivery system carrying a whole cell fraction, characterized in that the water-soluble and water-insoluble components of the whole cell are carried separately or together inside the particle and / or carried separately or together on the surface of the particle.

2. The targeted delivery system of claim 1 , wherein the targeting head is mannose.

3. The targeted delivery system of claim 1, wherein the specific cells or tissues are one or more of dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, basophils, lymph nodes, thymus, spleen, and bone marrow.

4. The targeted delivery system of claim 1, wherein the nanoscale size particles have a particle size of 1 nm to 1000 nm, and the microscale size particles have a particle size of 1 μm to 1000 μm.

5. The targeted delivery system of claim 1 or 4, wherein the manufacturing materials of the nanoscale-sized particles or microscale-sized particles are one or more of organic synthetic polymer materials, natural polymer materials, inorganic materials, bacteria or viruses.

6. The targeted delivery system of claim 5, wherein the organic synthetic polymer material is PLGA, PLA, PGA, poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, the natural polymer material is phospholipid, cholesterol, starch, sugar, polypeptide, sodium alginate, albumin, collagen, gelatin, cell membrane component, and the inorganic material is ferric oxide, ferric oxide, calcium carbonate, calcium phosphate.

7. The targeted delivery system of claim 1 , characterized in that the shape is spherical, elliptical, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangular, butterfly-shaped or disc-shaped.

8. The targeted delivery system of claim 1 further comprising an immune adjuvant.

9. The immune adjuvants include pattern recognition receptor agonists, Bacillus Calmette-Guerin (BCG) vaccine, immune adjuvant CpG, immune adjuvant poly(I:C), 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, polysaccharides, curcumin, immune adjuvant poly ICLC, Corynebacterium parvum 9. The targeted delivery system of claim 8, characterized in that the active ingredient is one or more of: 1) glycerol, ...

10. The targeted delivery system of claim 9, characterized in that the herbal medicine is ginseng (Ginseng) and astragalus.

11. The targeted delivery system according to any one of claims 1 to 10, further comprising adding a PEG protective film to the exterior of the particle.

12. After dissolving the cancer cells or tissues, first obtain the water-soluble components soluble in pure water or an aqueous solution without a solubilizing agent, and then dissolve the water-insoluble components in the solubilizing agent by using a solubilizing aqueous solution containing a specific solubilizing agent, thereby loading the whole cell fraction inside and / or on the surface of nanoparticles or microparticles to prepare a targeted delivery system; or The targeted delivery system according to any one of claims 1 to 11, characterized in that after lysing cells or tissues, the whole cell fraction is lysed directly using a solubilizing aqueous solution containing a solubilizing agent without separating the water-soluble and water-insoluble components, and the whole cell fraction dissolved in the solubilizing aqueous solution is used to manufacture the targeted delivery system.

13. A vaccine comprising the targeted delivery system of any one of claims 1 to 12.

14. A pharmaceutical composition comprising the targeted delivery system according to any one of claims 1 to 12 or the vaccine according to claim 13.

15. A vaccine according to claim 13, a targeted delivery system according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 14 for use in the prevention and / or treatment of cancer.

16. The vaccine, targeted delivery system or pharmaceutical composition for use according to claim 15, characterized in that the cancer is a solid tumor or a blood system tumor, including an endocrine system tumor, a nervous system tumor, a reproductive system tumor, a digestive system tumor, a urinary system tumor, an immune system tumor, a circulatory system tumor, a respiratory system tumor, a blood system tumor, and a skin tumor.

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