A dendritic cell cancer vaccine and its application

Abstract

The present invention relates to a dendritic cell cancer vaccine, which is obtained by activating dendritic cells in vitro with delivery particles loaded with cell components; wherein the delivery particles are nanoparticles and / or micron particles, the cell components are derived from water-soluble components and / or water-insoluble components of cells in cancer cells and / or tumor tissues, and the activation is to co-incubate the delivery particles loaded with cell components with dendritic cells. The present invention maximizes the types of antigens phagocytosed and presented by dendritic cells in vitro, and ensures stable therapeutic effects by selecting a suitable activation method.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and in particular to a dendritic cell cancer vaccine and application thereof. Background Art

[0002] Immunity is a physiological function of the human body. The human body relies on this function to identify "self" and "non-self" components, thereby destroying and removing abnormal substances (such as viruses, bacteria, etc.) in the human body, or damaged cells and tumor cells produced by the human body itself, in order to maintain human health. In recent years, immune technology has developed rapidly, especially in the field of cancer immunotherapy. With the continuous improvement of the understanding of cancer, people have found that the human immune system and various immune cells play a key role in inhibiting the occurrence and development of cancer. By regulating the balance of the body's immune system, we hope to influence and control the occurrence, development and treatment of cancer.

[0003] Cancer vaccines are one of the important methods in cancer immunotherapy and prevention. Dendritic cells (DCs) are the most important antigen presenting cells and the main cells that activate characteristic specific immune responses. Dendritic cells originate from bone marrow lymphocytes and can settle in tissues throughout the body to monitor the surrounding environment and transmit captured information to the adaptive immune system (T lymphocytes (hereinafter referred to as T cells) and B lymphocytes (hereinafter referred to as B cells) at any time. They are professional antigen presenting cells that express major histocompatibility complex (MHC) class I and II molecules and are the key link between innate immunity and adaptive immunity. Dendritic cells internalize and decompose antigens obtained from the periphery into short peptides, which are expressed on the surface of dendritic cells in the form of peptide-MHC complexes. This process is also the process of dendritic cell maturation. Then, dendritic cells loaded with antigen peptides migrate to secondary lymphoid organs to activate T cells. Compared with other antigen presenting cells, dendritic cells have extremely high antigen presentation efficiency and can induce very few T cell responses, becoming the most effective endogenous stimulus for T cell and B cell responses.

[0004] Dendritic cell cancer vaccine is a type of cancer vaccine. At present, the development of dendritic cancer vaccine mainly selects several polypeptide antigens or several protein antigens or the supernatant of cancer cell lysate to activate dendritic cells in vitro, and then infuses them into the body. At present, studies have shown that dendritic cells around tumors can capture tumor antigens released by tumor cells. These antigens come from dead tumor cells or live tumor cells engulfed by dendritic cells. These antigens are then cross-presented to the tumor to drain T cells in the lymph nodes, thereby inducing the production of tumor antigen-specific cytotoxic T cells (CTL) to kill tumor cells. However, in clinical practice, due to the overall immunosuppressive environment of the tumor and the interaction of multiple cells in the local immune microenvironment of the tumor, dendritic cells cannot successfully complete the immune response. In addition, the multiple molecules expressed and secreted by tumor cells in the tumor microenvironment can inhibit the activation of dendritic cells and drive the transformation of dendritic cells to inhibitory or regulatory phenotypes, thereby inhibiting the immune response of the tumor. Therefore, even if dendritic cell vaccines have a certain effect, not all clinical trials show that dendritic cell vaccines can benefit the survival of tumor patients. For example, Walker et al. conducted a phase I clinical trial that included 9 patients with glioblastoma and 4 patients with anaplastic astrocytoma. They were given dendritic cell vaccines combined with standard glioma treatment. After reoperation, T cell infiltration in tumor specimens increased, but overall survival was not significantly prolonged. In 2010, the U.S. Food and Drug Administration (FDA) approved the first dendritic cell vaccine, Provenge, for the treatment of refractory prostate cancer. However, since Provenge vaccine only used a limited number of antigens to stimulate and activate dendritic cells in vitro during its preparation, its clinical efficacy is very limited. Since then, dendritic cell vaccines have achieved certain efficacy in the treatment of breast cancer, bladder cancer, kidney cancer, colon cancer and rectal cancer, lung cancer, and melanoma, but they only activate dendritic cells through some antigens, and the therapeutic effect still needs to be improved.

[0005] The basis of cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormal mutated cancer cells. Cancer cells and tumor tissues are highly heterogeneous and have many mutations, so cancer cells or cancer tumor tissues themselves are the best sources of cancer antigens. The more dendritic cells there are, the more antigens they can engulf, and the better the efficacy of the vaccine. However, directly mixing tumor lysate with dendritic cells for incubation is difficult to be engulfed and taken up by dendritic cells because the cell membrane is fat-soluble and the components in the supernatant of the cell lysate used by previous researchers are water-soluble. Therefore, a new method for preparing dendritic cell vaccines is still needed. Summary of the invention

[0006] To solve the above technical problems, the present invention provides a dendritic cell vaccine, which activates dendritic cells in vitro based on micro- or nanoparticles loaded with whole cell components of one or more cancer cells and / or one or more tumor tissues or a mixture thereof.

[0007] The present invention discloses a dendritic cell cancer vaccine, which is obtained by activating dendritic cells in vitro with delivery particles loaded with cell components, wherein the delivery particles are nanoparticles and / or micron particles, the cell components are derived from water-soluble components and / or water-insoluble components of cells in one or more cancer cells and / or one or more tumor tissues, and the activation refers to co-incubating the delivery particles loaded with cell components with dendritic cells.

[0008] In the present invention, the cellular components are derived from components obtained from one or more cancer cells and / or one or more whole cells of tumor tissues. The water-insoluble components are loaded onto the delivery particles so that the vaccine system contains more antigens. More preferably, the water-soluble components and the water-insoluble components are loaded onto the delivery particles at the same time so that the delivery particles are loaded with all antigens. The particles are then co-incubated with dendritic cells in vitro, and after being phagocytosed by the dendritic cells, they can be presented and activated by the dendritic cells. After being infused back into the body, they can home to the lymph nodes and activate cancer-specific T cells using the antigens loaded by the DC cells.

[0009] Furthermore, the dendritic cells are autologous dendritic cells and / or allogeneic dendritic cells. The present invention uses in vitro activation of dendritic cells instead of in vivo activation because the inventors have found through a large number of experiments that due to the different environments of in vivo activation and in vitro activation, the presentation function of DC cells may not be exerted in vivo, resulting in differences in therapeutic effects.

[0010] Furthermore, the dendritic cells are derived from any cells that can be used to prepare and isolate dendritic cells, including but not limited to stem cells, bone marrow cells, and peripheral immune cells.

[0011] Furthermore, the delivery particles and the cell components loaded thereon are co-incubated with the dendritic cells for at least 4 hours, so that the cell components loaded by the micro-nanoparticles can be delivered to the dendritic cells and can be processed and presented by the dendritic cells. In the following embodiments, the co-incubation time is at least 4 hours, preferably 48-96 hours.

[0012] Furthermore, the water-soluble component in the present invention is the original water-soluble part of the cell or tissue that is soluble in pure water or an aqueous solution without a solubilizer, and the water-insoluble component is the original water-insoluble part of the cell or tissue that is changed from being insoluble in pure water to being soluble in an aqueous solution containing a solubilizer or an organic solvent by using an appropriate solubilization method. Among them, the cell component is obtained by lysing the whole cells of one or more cancer cells and / or tumor tissues, or by processing after lysing the whole cells of one or more cancer cells and / or tumor tissues, or by processing and lysing the whole cells of one or more cancer cells and / or tumor tissues.

[0013] Furthermore, both the water-soluble part and the water-insoluble part in the cell component can be dissolved by a solubilizing aqueous solution or an organic solvent containing a solubilizing agent. The solubilizing agent is at least one of the solubilizing agents that can increase the solubility of the protein or polypeptide in the aqueous solution; and the organic solvent is an organic solvent that can dissolve the protein or polypeptide.

[0014] Further, the solubilizing agent includes, but is not limited to, urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, alkaline solutions with a pH greater than 7, acidic solutions with a pH less than 7, various protein degrading enzymes, albumin, lecithin, high concentration inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, CA-630. It will be appreciated by those skilled in the art that the water-insoluble component may also be made soluble from insoluble in pure water by other methods that can solubilize proteins and polypeptide fragments. Organic solvents include, but are not limited to, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, and ethyl acetate. It will be appreciated by those skilled in the art that the organic solvent may also be made soluble by other methods that can solubilize proteins and polypeptide fragments by containing organic solvents.

[0015] Furthermore, the nanoparticle and / or microparticle system for activating dendritic cells includes nanoscale or microscale particles and cellular components loaded by the particles or a mixture thereof, wherein the mixture includes but is not limited to water-soluble components mixed with each other, or water-insoluble components mixed with each other, or all or part of the water-soluble components mixed with all or part of the water-soluble components.

[0016] Further, the cell components or a mixture thereof are loaded inside and / or on the surface of the micro-nano particles. Specifically, the loading method is that the water-soluble components and the water-insoluble components of the cells are separately or simultaneously encapsulated inside the particles, and / or separately or simultaneously loaded on the surface of the particles, including but not limited to the water-soluble components being loaded in the particles and loaded on the surface of the particles at the same time, the water-insoluble components being loaded in the particles and loaded on the surface of the particles at the same time, the water-soluble components being loaded in the particles and the water-insoluble components being loaded on the surface of the particles, the water-soluble components being loaded in the particles and the water-soluble components being loaded on the surface of the particles, the water-insoluble components being loaded in the particles and the water-soluble components being loaded on the surface of the particles, and the water-insoluble components being loaded in the particles and the water-soluble components being loaded on the surface of the particles. The water-soluble components and the water-insoluble components are loaded in the particles and only the water-soluble components are loaded on the particle surface, the water-soluble components and the water-insoluble components are loaded in the particles and only the water-soluble components are loaded on the particle surface, the water-soluble components are loaded in the particles and the water-soluble components and the water-insoluble components are loaded on the particle surface at the same time, the water-insoluble components are loaded in the particles and the water-soluble components and the water-insoluble components are loaded on the particle surface at the same time, the water-soluble components and the water-insoluble components are loaded in the particles at the same time and the water-soluble components and the water-insoluble components are loaded on the particle surface at the same time.

[0017] Furthermore, the micro-nanoparticles used to activate dendritic cells also include immunopotentiating adjuvants inside and / or on their surfaces. Immunopotentiating adjuvants include, but are not limited to, immunopotentiators of microbial origin, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicine and at least one of other categories; immunopotentiating adjuvants include, but are not limited to, pattern recognition receptor agonists, BCG (BCG), manganese-related adjuvants, BCG cell wall skeletons, BCG methanol extraction residues, BCG cell wall acyl dipeptide, Mycobacterium phlei, polyantigen A, mineral oil, virus-like particles, immunopotentiating reconstructed influenza virus bodies, cholera enterotoxin, saponin and its derivatives, Resiquimod, thymosin, newborn bovine liver active peptide, Miquimod, polysaccharides, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), immune adjuvant poly At least one of the effective ingredients of ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, manganese adjuvant, aluminum adjuvant, calcium adjuvant, various cytokines, interleukins, interferons, polyinosinic acid, polyadenylic acid, alum, aluminum phosphate, lanolin, squalene, cytokines, vegetable oils, endotoxins, liposome adjuvants, MF59, double-stranded RNA, double-stranded DNA, aluminum-related adjuvants, CAF01, ginseng, and astragalus. Those skilled in the art will appreciate that the enumeration here is not exhaustive, and the immune enhancing adjuvant may also use other substances that can enhance the immune response.

[0018] Furthermore, the immune adjuvant and the cell components are co-loaded into nanoparticles or microparticles. After the nanoparticles or microparticles are phagocytosed by dendritic cells, the dendritic cells can be better activated, so that the dendritic cells can play the role of preventing or treating cancer after being reinfused into the body.

[0019] Furthermore, the surface of the micro-nanoparticles may not be connected to a target head with active targeting function, or may be connected to a target head with active targeting function, which may be a commonly used target head such as mannose, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc., leading the particle system to be targeted and delivered into dendritic cells.

[0020] Furthermore, the micro-nanoparticle system loaded with cell components can be prepared by existing preparation methods, including but not limited to common solvent evaporation method, dialysis method, extrusion method, hot melt method. In some embodiments, the system is prepared by the double emulsion method in the solvent evaporation method.

[0021] Furthermore, nanoparticles and / or microparticles (nanoparticles and / or microparticles in the present invention are referred to as micro-nanoparticles for short) may not be modified during the preparation process, or appropriate modification techniques may be used to increase the antigen loading capacity and / or immunogenicity of the nanovaccine or microvaccine and thereby improve the efficacy of the dendritic cell vaccine.

[0022] Furthermore, the form in which the cell components or the mixture thereof are loaded inside the nano- and / or micro-particles is any form in which the cell components or the mixture thereof can be loaded inside the nano- and / or micro-particles.

[0023] Furthermore, the cell components or mixtures thereof are loaded onto the surface of nano- and / or micro-particles by methods including, but not limited to, adsorption, covalent bonding, charge interaction (such as adding positively charged substances, adding negatively charged substances), hydrophobic interaction, one-step or multiple-step solidification, mineralization, encapsulation, and the like.

[0024] Furthermore, the water-soluble components and / or water-insoluble components loaded on the surface of the micro-nanoparticles are one or more layers after loading. When the vaccine surface is loaded with multiple layers of water-soluble components and / or water-insoluble components, there are modifiers between the layers.

[0025] Further, the particle size of the nanoparticles or micron particles is nanometer-level or micron-level, which can ensure that the vaccine is phagocytosed by antigen presenting cells, and in order to improve the phagocytosis efficiency, the particle size should be within an appropriate range. The particle size of the nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, and most preferably, the particle size is 100nm-600nm; the particle size of the micron particles is 1μm-1000μm, more preferably, the particle size is 1μm-100μm, more preferably, the particle size is 1μm-10μm, and most preferably, the particle size is 1μm-5μm.

[0026] Furthermore, the surface of the micro-nanoparticles may be electrically neutral, negatively charged, or positively charged.

[0027] Furthermore, the preparation materials of the micro-nanoparticles are organic synthetic polymer materials, natural polymer materials or inorganic materials. Among them, organic synthetic polymer materials are biocompatible or degradable polymer materials, including but not limited to polylactic acid-glycolic acid copolymer PLGA, PLA, PGA, Poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydride, PDON, PPDO, PMMA, polyamino acid, synthetic polypeptide, synthetic lipid; natural polymer materials are biocompatible or degradable polymer materials, including but not limited to lecithin, cholesterol, starch, lipids, sugars, polypeptides, sodium alginate, albumin, collagen, gelatin, cell membrane components; inorganic materials are materials without obvious biological toxicity, including but not limited to ferric oxide, ferric oxide, calcium carbonate, calcium phosphate, etc.

[0028] Furthermore, the shape of the micro-nanoparticles is any common shape, including but not limited to spherical, ellipsoidal, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangle, butterfly-shaped or disc-shaped.

[0029] In some embodiments, the specific preparation method of the double emulsion method used in the present invention is as follows:

[0030] Step 1: adding a first predetermined volume of an aqueous solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of a medical polymer material.

[0031] In some embodiments, the aqueous solution may contain various components in the cancer cell lysate and immunoenhancing adjuvants poly(I:C), BCG, manganese adjuvant, calcium adjuvant or CpG; the various components in the cancer cell lysate are water-soluble components or original water-insoluble components dissolved in urea or guanidine hydrochloride during preparation. The concentration of water-soluble components from cancer cells contained in the aqueous solution or the concentration of original water-insoluble components from cancer cells dissolved in urea or guanidine hydrochloride, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 1 ng / mL, which can load enough cancer antigens to activate related immune responses. The concentration of the immunoenhancing adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0032] In some embodiments, the aqueous solution contains components of tumor tissue lysate and immunopotentiating adjuvants poly(I:C), BCG, manganese adjuvant, calcium adjuvant or CpG; each component of tumor tissue lysate is a water-soluble component or a originally water-insoluble component dissolved in urea or guanidine hydrochloride during preparation. The concentration of water-soluble components from tumor tissue contained in the aqueous solution or the concentration of originally water-insoluble components from tumor tissue dissolved in urea or guanidine hydrochloride, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01 ng / mL, which can load enough cancer antigens to activate related immune responses. The concentration of immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0033] In the present invention, the medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing a second predetermined concentration of the medical polymer material. In some embodiments, the medical polymer material is PLGA, and the organic solvent is dichloromethane. In addition, in some embodiments, the second predetermined concentration of the medical polymer material ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0034] In the present invention, PLGA or modified PLGA is selected because the material is a biodegradable material and has been approved by the FDA for use as a drug dressing. Studies have shown that PLGA has certain immunomodulatory functions and is therefore suitable as an auxiliary material for the preparation of nanoparticles or microparticles.

[0035] In practice, the second predetermined volume of the organic phase is set according to the ratio of the second predetermined volume of the organic phase to the first predetermined volume of the aqueous phase. In the present invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is in the range of 1:1.1-1:5000, preferably 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0036] Preferably, when the aqueous phase solution is a lysate component solution, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL; when the aqueous phase solution is a lysate component / immune adjuvant solution, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the polymer material organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion liquid is PBS buffer, physiological saline or pure water.

[0037] Step 2, subjecting the mixed solution obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization or microfluidic treatment for more than 1 minute. Preferably, when stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as a stirring speed of 50rpm to 1500rpm, and a stirring time of 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5W, and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure when using a high pressure / ultra-high pressure homogenizer is greater than 5psi, such as 20psi to 100psi, and the speed when using a high shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; when using microfluidic treatment, the flow rate is greater than 0.01mL / min, such as 0.1mL / min-100mL / min. Ultrasound, stirring, homogenization or microfluidics are used for nano-sizing and / or micronization. The length of ultrasound time, stirring speed or homogenization pressure and time can control the size of the prepared micro-nanoparticles. Too large or too small will lead to changes in particle size.

[0038] Step 3, adding the mixture obtained after the treatment in step 2 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and performing an ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing a homogenization treatment or a microfluidic treatment. In this step, the mixture obtained in step 2 is added to the emulsifier aqueous solution and continues to be ultrasonicated or stirred for nano- or micronization. This step is for nano- or micronization. The length of the ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles or micron particles. Too long or too short will bring about changes in the particle size. For this reason, it is necessary to select a suitable ultrasonic time. In the present invention, the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds, the stirring speed is greater than 50rpm, such as 50rpm to 500rpm, and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as the stirring speed is 50rpm~1500rpm, and the stirring time is 0.5 hour~5 hours; during ultrasonic treatment, the ultrasonic power is 50W~500W, and the time is greater than 0.1 second, such as 2~200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure is greater than 20psi when using a high pressure / ultra-high pressure homogenizer, such as 20psi~100psi, and the speed is greater than 1000rpm when using a high shear homogenizer, such as 1000rpm~5000rpm; the flow rate of microfluidic treatment is greater than 0.01mL / min, such as 0.1mL / min-100mL / min. Ultrasound or stirring or homogenization or microfluidic treatment is carried out for nano- or micronization, and the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared nano- or micron particles, and too large or too small will bring about changes in particle size.

[0039] In the present invention, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg / mL. The third predetermined volume is adjusted according to its ratio with the second predetermined volume. In the present invention, the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5. In order to control the size of nanoparticles or micron particles during specific implementation, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time of this step, the volume of the emulsifier aqueous solution and the concentration are based on the values ​​of all in order to obtain nanoparticles or micron particles of suitable size.

[0040] Step 4, adding the liquid obtained after the treatment in step 3 to a fourth predetermined volume of an aqueous emulsifier solution of a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.

[0041] In this step, the emulsifier aqueous solution is still PVA.

[0042] The fourth predetermined concentration is 5 mg / mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles or micron particles of suitable size. The selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or micron particles.

[0043] In the present invention, the predetermined stirring condition of this step is until the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized.

[0044] Step 5, after the mixed solution that meets the predetermined stirring conditions treated in step 4 is centrifuged at a speed greater than 100 RPM for more than 1 minute, the supernatant is removed, and the remaining precipitate is re-suspended in a fifth predetermined volume of an aqueous solution containing a lyophilized protective agent at a fifth predetermined concentration or in a sixth predetermined volume of PBS (or physiological saline).

[0045] In some embodiments of the present invention, the precipitate obtained in step 5 does not need to be freeze-dried when it is resuspended in the sixth predetermined volume of PBS (or physiological saline), and can be directly used for subsequent experiments related to the adsorption of cancer cell lysates on the surface of nanoparticles or microparticles.

[0046] In some embodiments of the present invention, the precipitate obtained in step 5 needs to be freeze-dried when it is re-suspended in an aqueous solution containing a lyoprotectant, and then subsequent experiments related to adsorption of cancer cell lysates on the surface of nanoparticles or microparticles are performed after freeze-drying.

[0047] In the present invention, the lyophilization protective agent is trehalose.

[0048] In the present invention, the fifth predetermined concentration of the lyophilization protectant in this step is 4% by mass, which is set in order not to affect the lyophilization effect in the subsequent freeze-drying.

[0049] Step 6, freeze-drying the suspension containing the lyophilization protectant obtained in step 5, and then keeping the lyophilized material for later use.

[0050] Step 7, the nanoparticle-containing suspension obtained in step 5 and resuspended in PBS (or physiological saline) with a sixth predetermined volume, or the freeze-dried material containing nanoparticles or microparticles and lyoprotectant obtained in step 6 and resuspended in a sixth predetermined volume of PBS (or physiological saline), is mixed with a seventh predetermined volume of water-soluble components or the original water-insoluble components dissolved in 8M urea to obtain a nanoparticle or microparticle system.

[0051] In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.

[0052] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble components in the cancer cell lysate or tumor tissue lysate or the original water-insoluble components dissolved in 8M urea is 1 mL. In actual use, the volumes and ratios of the two can be adjusted as needed.

[0053] Step 8, mixing the nanoparticles and / or microparticles prepared in step 7 with dendritic cells and incubating them for a certain period of time.

[0054] Step 9, collecting the dendritic cells activated in step 8 and returning them to the body to prevent or treat cancer.

[0055] In other embodiments, the specific preparation method of the double emulsion method adopted by the present invention is as follows:

[0056] Step 1: adding a first predetermined volume of an aqueous solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of a medical polymer material.

[0057] In some embodiments, the aqueous solution may contain various components in the cancer cell lysate and immunoenhancing adjuvants poly(I:C), manganese adjuvants, calcium adjuvants, BCG or CpG; the various components in the cancer cell lysate are water-soluble components or original water-insoluble components dissolved in urea or guanidine hydrochloride during preparation. The concentration of water-soluble components from cancer cells contained in the aqueous solution or the concentration of original water-insoluble components from cancer cells dissolved in urea or guanidine hydrochloride, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01 ng / mL, which can load enough cancer antigens to activate related immune responses. The concentration of the immunoenhancing adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0058] In some embodiments, the aqueous solution contains components of the tumor tissue lysate and immunoenhancing adjuvants poly(I:C), manganese adjuvant, calcium adjuvant, BCG or CpG; each component of the tumor tissue lysate is a water-soluble component or a originally water-insoluble component dissolved in urea or guanidine hydrochloride during preparation. The concentration of the water-soluble component from the tumor tissue contained in the aqueous solution or the concentration of the original water-insoluble component from the tumor tissue dissolved in urea or guanidine hydrochloride, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01 ng / mL, which can load enough cancer antigens to activate related immune responses. The concentration of the immunoenhancing adjuvant in the initial aqueous phase is greater than 0.01 ng / mL.

[0059] In the present invention, the medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing a second predetermined concentration of the medical polymer material. In some embodiments, the medical polymer material is PLGA, and the organic solvent is dichloromethane. In addition, in some embodiments, the second predetermined concentration of the medical polymer material ranges from 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0060] In the present invention, PLGA or modified PLGA is selected because the material is a biodegradable material and has been approved by the FDA for use as a drug dressing. Studies have shown that PLGA has certain immunomodulatory functions and is therefore suitable as an auxiliary material for the preparation of nanoparticles or microparticles.

[0061] In practice, the second predetermined volume of the organic phase is set according to the ratio of the second predetermined volume of the organic phase to the first predetermined volume of the aqueous phase. In the present invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is in the range of 1:1.1-1:5000, preferably 1:10. In the specific implementation process, the first predetermined volume, the second predetermined volume and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0062] Preferably, when the aqueous phase solution is a lysate component solution, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL; when the aqueous phase solution is a lysate component / immune adjuvant solution, the concentration of proteins and polypeptides therein is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immune adjuvant is greater than 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the polymer material organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the polymer material is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL. The first emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 10 mg / mL to 50 mg / mL, preferably 20 mg / mL. The second emulsifier solution is preferably a polyvinyl alcohol aqueous solution, with a concentration of 1 mg / mL to 20 mg / mL, preferably 5 mg / mL. The dispersion liquid is PBS buffer or physiological saline or pure water.

[0063] Step 2, subjecting the mixed solution obtained in step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization or microfluidic treatment for more than 1 minute. Preferably, when stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as a stirring speed of 50rpm to 1500rpm, and a stirring time of 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5W, and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure when using a high pressure / ultra-high pressure homogenizer is greater than 5psi, such as 20psi to 100psi, and the speed when using a high shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; when using microfluidic treatment, the flow rate is greater than 0.01mL / min, such as 0.1mL / min-100mL / min. Ultrasound, stirring, homogenization or microfluidics are used for nano-sizing and / or micronization. The length of ultrasound time, stirring speed or homogenization pressure and time can control the size of the prepared micro-nanoparticles. Too large or too small will lead to changes in particle size.

[0064] Step 3, adding the mixture obtained after the treatment in step 2 to a third predetermined volume of an aqueous solution containing a third predetermined concentration of an emulsifier and performing an ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing a homogenization treatment or a microfluidic treatment. In this step, the mixture obtained in step 2 is added to the emulsifier aqueous solution and continues to be ultrasonicated or stirred for nano- or micronization. This step is for nano- or micronization. The length of the ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles or micron particles. Too long or too short will bring about changes in the particle size. For this reason, it is necessary to select a suitable ultrasonic time. In the present invention, the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds, the stirring speed is greater than 50rpm, such as 50rpm to 500rpm, and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, such as the stirring speed is 50rpm~1500rpm, and the stirring time is 0.5 hour~5 hours; during ultrasonic treatment, the ultrasonic power is 50W~500W, and the time is greater than 0.1 second, such as 2~200 seconds; during homogenization, a high pressure / ultra-high pressure homogenizer or a high shear homogenizer is used, and the pressure is greater than 20psi when using a high pressure / ultra-high pressure homogenizer, such as 20psi~100psi, and the speed is greater than 1000rpm when using a high shear homogenizer, such as 1000rpm~5000rpm; the flow rate of microfluidic treatment is greater than 0.01mL / min, such as 0.1mL / min-100mL / min. Ultrasound or stirring or homogenization or microfluidic treatment is carried out for nano- or micronization, and the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared nano- or micron particles, and too large or too small will bring about changes in particle size.

[0065] In the present invention, the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20mg / mL. The third predetermined volume is adjusted according to its ratio with the second predetermined volume. In the present invention, the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5. In order to control the size of nanoparticles or micron particles during specific implementation, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the ultrasonic time or stirring time of this step, the volume of the emulsifier aqueous solution and the concentration are based on the values ​​of all in order to obtain nanoparticles or micron particles of suitable size.

[0066] Step 4, adding the liquid obtained after the treatment in step 3 to a fourth predetermined volume of an aqueous emulsifier solution of a fourth predetermined concentration, and stirring until a predetermined stirring condition is met, or directly performing subsequent treatment without stirring.

[0067] In this step, the emulsifier aqueous solution is still PVA.

[0068] The fourth predetermined concentration is 5 mg / mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles or micron particles of suitable size. The selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. In the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or micron particles.

[0069] In the present invention, the predetermined stirring condition of this step is that the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized. Subsequent tests are not performed without stirring.

[0070] Step 5, after centrifuging the mixed solution that meets the predetermined stirring conditions in step 4 at a rotation speed greater than 100 RPM for more than 1 minute, remove the supernatant and re-suspend the remaining precipitate in a fifth predetermined volume of a solution of a fifth predetermined concentration containing water-soluble and / or water-insoluble components in the whole cell component, or re-suspend the remaining precipitate in a fifth predetermined volume of a solution of a fifth predetermined concentration containing water-soluble and / or water-insoluble components in the whole cell component and an adjuvant.

[0071] Step 6, after the mixed solution that meets the predetermined stirring conditions in step 5 is centrifuged at a speed greater than 100 RPM for more than 1 minute, the supernatant is removed, and the remaining precipitate is re-suspended in the sixth predetermined volume of solidification treatment reagent or mineralization treatment reagent, centrifuged and washed after a certain period of time, and then the seventh predetermined volume of positively or negatively charged substances is added and allowed to react for a certain period of time.

[0072] In some embodiments of the present invention, the precipitate obtained in step 6 may not need to be freeze-dried after being resuspended in the seventh predetermined volume of charged substance, and subsequent experiments related to loading cancer cell / tissue lysate on the surface of nanoparticles or microparticles may be directly performed.

[0073] In some embodiments of the present invention, the precipitate obtained in step 6 is resuspended in an aqueous solution containing a drying protectant and then vacuum dried at room temperature or freeze-dried in a vacuum. After drying, subsequent experiments related to adsorption of cancer cell lysates on the surface of nanoparticles or microparticles are performed.

[0074] In the present invention, the freeze-drying protective agent is selected from trehalose or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the freeze-drying protective agent in this step is 4% by mass, which is set so as not to affect the drying effect in the subsequent drying.

[0075] Step 7, drying the suspension containing the drying protective agent obtained in step 6, and keeping the dried material for later use.

[0076] Step 8, the suspension containing nanoparticles obtained in step 6 and resuspended in PBS (or physiological saline) with an eighth predetermined volume, or the dried material containing nanoparticles or micron particles and a drying protective agent obtained in step 7 and resuspended in an eighth predetermined volume of PBS (or physiological saline), is mixed with a ninth predetermined volume of water-soluble components or water-insoluble components to obtain nanoparticles or micron particles.

[0077] In the present invention, the modification and antigen loading steps of step 5 to step 8 can be repeated multiple times to increase the antigen loading amount. Moreover, when adding positively or negatively charged substances, substances with the same charge can be added multiple times or substances with different charges can be added alternately.

[0078] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble components or the original water-insoluble components in the cancer cell lysate or tumor tissue lysate is 0.1-100 mL. In actual use, the volumes and ratios of the two can be adjusted as needed.

[0079] Step 9, mixing the nanoparticles and / or microparticles prepared in step 8 with dendritic cells and incubating them for a certain period of time.

[0080] Step 10, collecting the dendritic cells activated in step 9 and returning them to the body to prevent or treat cancer.

[0081] In the present invention, the water-soluble component or the original water-insoluble component of the cancer cell lysate or tumor tissue lysate contains poly(I:C), manganese adjuvant, BCG (BCG) or CpG, and the concentration of poly(I:C), calcium adjuvant, BCG or CpG is greater than 0.01 ng / mL.

[0082] Furthermore, when preparing the vaccine of the present invention, when activating dendritic cells in vitro, nanoparticles and / or microparticles that only load water-soluble components and nanoparticles and / or microparticles that only load water-insoluble components can be used simultaneously, nanoparticles and / or microparticles that only load water-soluble components can be used, nanoparticles and / or microparticles that only load water-insoluble components can be used, or nanoparticles and / or microparticles that simultaneously load water-soluble components and water-insoluble components can be used.

[0083] From the above technical scheme, it can be known that the present invention provides a delivery system for delivering water-soluble components and / or water-insoluble components of cells using nano-sized or micron-sized particles, and uses the above micro-nano particle system to activate dendritic cells in vitro for the prevention and treatment of cancer. Because the cell components of the relevant cells or tissues are divided into two parts according to their solubility in pure water, a water-soluble part soluble in pure water and a water-insoluble part insoluble in pure water, and both the water-soluble part and the water-insoluble part are loaded in the micro-nano particles, most of the mutated proteins or polypeptides produced by cancer in the cell components are loaded in the micro-nano particles for in vitro activation of dendritic cells. The water-soluble part and the water-insoluble part in the cell components include the components of the entire cell; the water-soluble part and the water-insoluble part in the cell components can also be dissolved by an aqueous solution containing a solubilizing agent at the same time, wherein the proteins, polypeptides and genes that are the same as normal cell components and have not mutated will not cause an immune response because of the immune tolerance generated during the development of the autoimmune system; and the mutations of genes, proteins and polypeptides produced by cancer, etc., are immunogenic and can activate dendritic cells because there is no 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 activate dendritic cells for the prevention, treatment and recurrence of cancer.

[0084] Furthermore, at least one of the cancer cells or tumor tissues is the same as the target disease type.

[0085] Furthermore, when used as a cancer vaccine to prevent and treat cancer, the vaccine of the present invention can be administered multiple times before or after cancer occurs or after surgical resection of tumor tissue to activate the body's immune system, thereby delaying the progression of cancer, treating cancer, or preventing the recurrence of cancer.

[0086] By means of the above scheme, the present invention has at least the following advantages:

[0087] The present invention provides a delivery system based on nano-sized or micron-sized particles to deliver water-soluble components and / or water-insoluble components of cells to activate dendritic cells in vitro for application in a vaccine system for preventing and treating cancer, thereby maximizing the types of antigens phagocytosed and presented by dendritic cells in vitro, and cancer-specific T cells activated by antigens in whole cell components or their mixtures can prevent or treat cancer.

[0088] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0090] Figure 1 Schematic diagram of the preparation process and application field of the vaccine system of the present invention; wherein, a is a schematic diagram of collecting and preparing nanoparticles or micron particles from water-soluble components and water-insoluble components respectively; b is a schematic diagram of using a solubilizing solution containing a solubilizing agent to dissolve whole cell components and prepare nanoparticles or micron particles; c is a schematic diagram of using the above particles prepared in a or b to activate dendritic cell vaccines and use the vaccine to prevent or treat cancer;

[0091] Figure 2-13 The results of the experiments on tumor growth rate and survival period of mice when dendritic cell vaccines were used to prevent or treat cancer in Examples 1-12 are as follows: a, the experimental results on tumor growth rate when vaccines were used to prevent or treat cancer (n≥8); b, the experimental results on survival period of mice when vaccines were used to prevent or treat cancer (n≥8), each data point is mean±standard error (mean±SEM); the significant differences in the tumor growth inhibition experiment in Figure a were analyzed by ANOVA, and the significant differences in Figure b were analyzed by Kaplan-Meier and log-rank test analysis; *** indicates that the vaccine group was significantly different from the PBS blank control group (p < 0.005); ### indicates that the blank nanoparticle-activated dendritic cell control group was significantly different from the control group (p < 0.005); & indicates that the vaccine group was significantly different from the lysate-directly activated dendritic cell control group (p < 0.05); && indicates that the vaccine group was significantly different from the lysate-directly activated dendritic cell control group (p < 0.01); &&& indicates that the vaccine group was significantly different from the lysate-directly activated dendritic cell control group (p < 0.005); $ indicates that the modified nanoparticle-activated dendritic cell vaccine group was significantly different from the unmodified nanoparticle-activated dendritic cell vaccine group (p < 0.05). Difference; $$ represents the difference between the modified nanoparticle-activated dendritic cell vaccine group and the unmodified nanoparticle-activated dendritic cell vaccine group, p < 0.01, which is significant; ★ represents the difference between the dendritic cell vaccine group activated by nanoparticles loaded with whole cell components and the dendritic cell vaccine group activated by nanoparticles loaded with multiple polypeptide neoantigens, p < 0.05, which is significant; δ represents the difference between the dendritic cell vaccine group activated by nanoparticles loaded with both cell components and adjuvants and the dendritic cell vaccine group activated by nanoparticles loaded with only cell components, p < 0.05, which is significant; τ represents the difference between the dendritic cell vaccine group activated by nanoparticles with target heads and the dendritic cell vaccine group activated by nanoparticles without target heads, p < 0.05, which is significant. DETAILED DESCRIPTION

[0092] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0093] The delivery system of the whole cell components or the mixture thereof of the present invention can be used to activate dendritic cells in vitro to prepare dendritic cell vaccines for preventing and / or treating cancer. The preparation process and application fields thereof are as follows: Figure 1 During the preparation, the cells or tissues can be lysed, and then the water-soluble components and the water-insoluble components can be collected separately to prepare the nano- or micro-particle system respectively; or the cells or tissues can be directly lysed using a solubilizing solution containing a solubilizing agent, and the whole cell components can be dissolved to prepare the nano- or micro-particle system.

[0094] The whole cell components of the present invention can be treated before or after lysis, including but not limited to inactivation or denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc., and then the nano-vaccine or micro-vaccine can be prepared; or the nano-vaccine or micro-vaccine can be directly prepared without any inactivation or denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment before or after cell lysis. In some embodiments of the present invention, tumor tissue cells are inactivated or denatured before lysis, and in actual use, they can also be inactivated or denatured after cell lysis, or inactivated or denatured before and after cell lysis; in some embodiments of the present invention, the inactivation or denaturation treatment method before or after cell lysis is ultraviolet irradiation and high temperature heating, and in actual use, treatment methods including but not limited to radiation irradiation, high pressure, solidification, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze drying, etc. can also be used. Those skilled in the art will appreciate that in actual application, the technicians may make appropriate adjustments according to specific circumstances.

[0095] Example 1 Nanoparticles loaded with whole cell components of tumor tissue activated dendritic cells in vitro and then infused for the treatment of melanoma

[0096] This example uses mouse melanoma as a cancer model to illustrate how to use a nanoparticle system loaded with whole cell components of melanoma tumor tissue to activate dendritic cells in vitro and then infuse the dendritic cells back into mice to treat melanoma. In this example, B16F10 melanoma tumor tissue is first lysed to prepare water-soluble components and water-insoluble components of the tumor tissue, then, an organic polymer material PLGA is used as a nanoparticle skeleton material, Polyinosinic-polycytidylic acid (poly(I:C)) is used as an immune adjuvant, and a solvent volatilization method is used to prepare a nanoparticle system loaded with water-soluble components and water-insoluble components of the tumor tissue, and then the nanoparticle system is co-incubated with dendritic cells (DC) in vitro and then the dendritic cells are infused back into the body to treat melanoma.

[0097] (1) Lysis of tumor tissue and collection of various components

[0098] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16-F10 cells, when the tumor grows to a volume of approximately 1000mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were cut into pieces and ground, and an appropriate amount of pure water was added through a cell filter and repeatedly frozen and thawed 5 times, and ultrasound was used to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution. The above is the source of antigen raw materials for preparing the nanoparticle system.

[0099] (2) Preparation of nanoparticle systems

[0100] In this embodiment, the nano vaccine and the blank nanoparticles used as the control are prepared by the double emulsion method in the solvent evaporation method. During the preparation, the nano vaccine loaded with the water-soluble component in the whole cell component and the nanoparticles loaded with the water-insoluble component in the whole cell component are prepared separately, and then used together. The molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa, and the immune adjuvant used is poly(I:C) and poly(I:C) is only distributed inside the nanoparticles. The preparation method is as described above. In the preparation process, the double emulsion method is first used to load the antigen inside the nanoparticles. After the antigen (lysate component) is loaded inside, 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, and resuspended with 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h; before use, it is resuspended with 4mL PBS and then 1mL of tumor tissue lysate component (protein concentration 80mg / mL) is added and acted at room temperature for 10min to obtain a nanoparticle system with both internal and external lysates loaded. The average particle size of the nanoparticles is about 320nm, and the surface potential of the nanoparticles is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 160μg of protein or polypeptide components, and each 1mg of PLGA nanoparticles uses about 0.02mg of poly(I:C) immune adjuvant. The particle size of the blank nanoparticles is about 300nm. When preparing the blank nanoparticles, pure water or 8M urea containing an equal amount of poly(I:C) is used to replace the corresponding water-soluble components and water-insoluble components.

[0101] (3) Preparation of dendritic cells

[0102] This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDC). First, a 6-8 week old C57 mouse was killed by cervical dislocation, and the tibia and femur of the hind legs were surgically removed and placed in PBS, and the muscle tissue around the bone was removed cleanly with scissors and tweezers. Cut off the two ends of the bone with scissors, and then extract the PBS solution with a syringe, insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly flush the bone marrow into a culture dish. Collect the bone marrow solution, centrifuge at 400g for 3min, and then add 1mL of red blood cell lysis solution to lyse the red blood cells. Add 3mL of RPMI 1640 (10% FBS) culture medium to terminate the lysis, centrifuge at 400g for 3min, and discard the supernatant. The cells were placed in a 10mm culture dish and cultured, using RPMI 1640 (10% FBS) culture medium, and recombinant mouse GM-CSF (20ng / mL) was added at 37 degrees and 5% CO2 for 7 days. On the third day, the culture flask was gently shaken and supplemented with the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL). On the sixth day, half of the medium was replaced. On the seventh day, a small amount of suspended and semi-adherent cells were collected and detected by flow cytometry. +CD80 + CD11c + When the ratio of BMDC in cells is between 15-20%, the induced cultured BMDC can be used for the next experiment.

[0103] (4) Activation of dendritic cells

[0104] Mouse BMDCs were plated on a cell culture plate, and 5 mL of RPMI 1640 (10% FBS) medium was added to every 100,000 DC cells. Then, 30 μg of PLGA nanoparticles loaded with water-soluble components and 30 μg of PLGA nanoparticles loaded with water-insoluble components were added and incubated with BMDCs for 48 h. BMDCs were then collected and centrifuged at 300 g for 5 minutes, washed twice with phosphate buffered saline (PBS), and resuspended in PBS for later use.

[0105] (5) Dendritic cell cancer vaccines for cancer treatment

[0106] The control groups of this study were PBS group and blank nanoparticle-stimulated BMDC group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells. The dosing regimen for the dendritic cell vaccine group was as follows: 100 μL of vaccine containing 1 million dendritic cells was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after melanoma inoculation. The PBS control group regimen was as follows: 100 μL of PBS was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after melanoma inoculation. Blank nanoparticle control group: 100 μL of dendritic cells stimulated with 500,000 blank nanoparticles was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after melanoma inoculation. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0107] (4) Experimental results

[0108] like Figure 2 As shown, the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger, and the tumor growth rate of mice in the vaccine group was significantly slower than that in the control group, and some mice had no tumors and recovered. In summary, the dendritic cell vaccine of the present invention has a good therapeutic effect on melanoma.

[0109] Example 2 Tumor tissue whole cell components loaded on nanoparticles and activated in vitro dendritic cell vaccine for the prevention of melanoma

[0110] This example uses mouse melanoma as a cancer model to illustrate how to use dendritic cell vaccines to prevent cancer. In this example, B16F10 melanoma tumor tissue is first lysed to prepare water-soluble and water-insoluble components of the tumor tissue; then, a nanoparticle system loaded with water-soluble and water-insoluble components of the tumor tissue is prepared. In this example, the method of silicification and adding charged substances is used to increase the antigen loading, and only one round of mineralization treatment is performed.

[0111] (1) Lysis of tumor tissue and collection of various components

[0112] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16-F10 cells, when the tumor grows to a volume of approximately 1000mm 3 The mice were killed and the tumor tissue was removed. The tumor tissue was cut into pieces and ground, collagenase was added and incubated in RPMI1640 medium for 30 minutes, and then an appropriate amount of pure water was added through a cell filter and repeatedly frozen and thawed 5 times, and ultrasound was used to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed of 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into soluble in 8M urea aqueous solution. The above is the source of antigen raw materials for preparing particles.

[0113] (2) Preparation of Nanoparticles

[0114] Nanoparticle and the blank nanoparticle in the present embodiment adopt the multiple emulsion method preparation in the solvent evaporation method as a contrast, and the multiple emulsion method has been suitably modified and improved, and adopts low-temperature siliconization technology and two kinds of modification methods of adding charged substances to improve the antigenic load in the nanoparticle preparation process.The nanoparticle of the water-soluble component in the load full cell component and the nanoparticle of the water-insoluble component in the load full cell component are prepared respectively when preparing, and then used together when using.The nanoparticle preparation material PLGA molecular weight adopted is 24KDa-38KDa, and the immunoadjuvant adopted is poly (I: C) and poly (I: C) both distributed in the nanoparticle inside and also loaded on the nanoparticle surface. Preparation method As described above, in the preparation process, the antigen and adjuvant are first loaded inside the nanoparticles by the double emulsion method. After the antigen (lysis component) is loaded inside, 100 mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and then the nanoparticles are resuspended in 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (60 mg / mL), and then centrifuged at 10000g for 20 minutes, and then 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 5.0) is used. 3.0) and fixed at room temperature for 10 min, then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, resuspended in 3 mL of PBS containing protamine (5 mg / mL) and polylysine (10 mg / mL) for 10 min, then washed by centrifugation at 10000 g for 20 min, resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000 g for 20 min, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h; before use, the particles were resuspended in 7 mL of PBS and then 3 mL of cancer tissue lysate component containing adjuvant (protein concentration 50 mg / mL) was added and allowed to act at room temperature for 10 min, to obtain a nanoparticle system modified by cryosilicification and addition of cationic substances with lysate loaded both inside and outside. The average particle size of the nanoparticles is about 350nm, and the surface potential of the nanoparticles is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 300μg of protein or polypeptide components, and the poly(I:C) immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, with half inside and half outside.

[0115] The steps of the preparation method of the unmodified nanoparticles are basically the same as those of the modified nanoparticles, except that the steps of low-temperature silicification and adding charged substances are not performed. In the preparation process, the antigen is first loaded inside the nanoparticles by the double emulsion method, and after the antigen (lysate component) is loaded inside, it is centrifuged at 10000g for 20 minutes, and then resuspended with 10mL ultrapure water containing 4% trehalose and freeze-dried for 48h. Before the particles are used, they are resuspended with 7mL PBS and then 3mL cancer tissue lysate components (protein concentration 50mg / mL) containing adjuvants are added and acted at room temperature for 10min to obtain nanoparticles loaded with lysate inside and outside. The average particle size of the nanoparticles is about 320nm, and the surface potential of the nanoparticles is about -5mV; each 1mg PLGA nanoparticle is loaded with about 150μg protein or polypeptide components, and the poly (I:C) immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.02mg, and half is inside and half is outside.

[0116] The particle size of the blank nanoparticles is about 300 nm. When preparing the blank nanoparticles, pure water or 8 M urea containing an equal amount of poly(I:C) is used to replace the corresponding water-soluble components and water-insoluble components.

[0117] (3) Preparation of dendritic cells

[0118] This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDC). First, a 6-8 week old C57 mouse was killed by cervical dislocation, and the tibia and femur of the hind legs were surgically removed and placed in PBS, and the muscle tissue around the bone was removed cleanly with scissors and tweezers. Cut off the two ends of the bone with scissors, and then extract the PBS solution with a syringe, insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly flush the bone marrow into a culture dish. Collect the bone marrow solution, centrifuge at 400g for 3min, and then add 1mL of red blood cell lysis solution to lyse the red blood cells. Add 3mL of RPMI 1640 (10% FBS) culture medium to terminate the lysis, centrifuge at 400g for 3min, and discard the supernatant. The cells were placed in a 10mm culture dish and cultured, using RPMI 1640 (10% FBS) culture medium, and recombinant mouse GM-CSF (20ng / mL) was added at 37 degrees and 5% CO2 for 7 days. On the third day, the culture flask was gently shaken and supplemented with the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL). On the sixth day, half of the medium was replaced. On the seventh day, a small amount of suspended and semi-adherent cells were collected and detected by flow cytometry. + CD80 + When the proportion of cells in CD11c+ cells is between 15-20%, the induced cultured BMDCs can be used for the next experiment.

[0119] (4) Activation of dendritic cells

[0120] Mouse BMDCs were plated on a cell culture plate, and 5 mL of RPMI 1640 (10% FBS) medium was added to every 100,000 DC cells. Then, 20 μg of PLGA nanoparticles loaded with water-soluble components and 20 μg of PLGA nanoparticles loaded with water-insoluble components were added and incubated with BMDCs for 72 h. BMDCs were then collected and centrifuged at 300 g for 5 minutes, washed twice with PBS, and resuspended in PBS for later use.

[0121] (5) Dendritic cell vaccines for cancer prevention

[0122] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. The administration scheme of the dendritic cell vaccine group was as follows: 100 μL of dendritic cell vaccine (500,000 dendritic cells) was subcutaneously injected on days 35, 28, 21, 14, and 7 before melanoma inoculation; 1.5×10 5 B16F10 cells. The PBS control group was as follows: 100 μL PBS was injected subcutaneously on days 35, 28, 21, 14, and 7 before melanoma inoculation; 1.5×105 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse on day 0. Free lysate control group: 100 μL free lysate-activated dendritic cells were injected subcutaneously on days 35, 28, 21, 14, and 7 before melanoma inoculation; 1.5×105 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse on day 0. 5 In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0123] (6) Experimental results

[0124] like Figure 3 As shown, the tumors of mice in the control group grew, while the tumor growth rate of mice immunized with dendritic cell vaccines activated by nanoparticles loaded with antigens was significantly slowed down. Moreover, the dendritic cell vaccine activated by nanoparticles modified by siliconization and adding charged substances has a better preventive effect on melanoma than the nanoparticle-activated dendritic cell vaccine group that was not modified during the preparation process.

[0125] Example 3 Whole cell components of cancer cells loaded on nanoparticles to activate dendritic cells for cancer prevention

[0126] In this embodiment, B16F10 melanoma cancer cells were first lysed to prepare water-soluble components and water-insoluble components. Then, the nanoparticle system loaded with whole cell components of cancer cells was prepared using organic polymer material PLGA as nanoparticle skeleton material and CpG as immune adjuvant. In this embodiment, the method of siliconization, adding cationic substances and anionic substances was used to increase the antigen loading, and two rounds of siliconization were performed. After the nanoparticles were co-incubated with dendritic cells, the dendritic cells were re-infused to prevent cancer.

[0127] (1) Lysis of cancer cells and collection of components

[0128] After the cultured B16F10 melanoma cancer cell line is collected, it is centrifuged at 350g for 5 minutes, and then the supernatant is discarded and washed twice with PBS. Then the cells are resuspended in ultrapure water and repeatedly frozen and thawed 5 times, and ultrasound can be used to destroy the lysed cells. After the cells are lysed, the lysate is centrifuged at a speed of 3000g for 6 minutes and the supernatant is taken as the water-soluble component soluble in pure water; 8M urea is added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water can be converted into soluble in 8M urea aqueous solution. The above is the source of antigen raw materials for preparing the particle system.

[0129] (2) Preparation of nanoparticle systems

[0130] Nanoparticle and the blank nanoparticle in the present embodiment adopt the multiple emulsion method preparation in the solvent evaporation method as a contrast, and the multiple emulsion method has been suitably modified and improved, and adopts low-temperature siliconization technology and two kinds of modification methods of adding charged substances to improve the antigenic load in the nanoparticle preparation process.The nanoparticle of water-soluble components in the load full cell component and the nanoparticle of non-water-soluble components in the load full cell component are prepared respectively when preparing, and then used together when using.The nanoparticle preparation material PLGA molecular weight adopted is 7KDa-17KDa, and the immunoadjuvant adopted is that CpG and CpG both are distributed in the nanoparticle inside and are also loaded on the nanoparticle surface. The preparation method is as described above. During the preparation process, the antigen is first loaded inside the nanoparticles by the emulsion method. After the antigen (lysis component) is loaded inside, 100 mg of the nanoparticles is centrifuged at 10,000 g for 20 minutes, and then the nanoparticles are resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), and then centrifuged at 10,000 g for 20 minutes, and then resuspended with 10 mL of silicate solution (containing 120 mM NaCl, 100 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 12 hours, washed by centrifugation with ultrapure water, and then resuspended with 3 mL of PBS containing polyaspartic acid (10 mg / mL) and acted for 10 minutes, and then washed by centrifugation at 12,000 g for 18 minutes, and resuspended with 10 mL of PBS solution containing cell lysate (50 mg / mL) and acted for 10 minutes, and then centrifuged at 10,000 g for 20 minutes. Then, 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 3.0) was used and fixed at room temperature for 12 h. After centrifugation and washing with ultrapure water, 3 mL of PBS containing histone (5 mg / mL) and polyarginine (10 mg / mL) was used to resuspend and act for 10 min, and then centrifuged at 10000 g for 20 min to wash, and 10 mL of PBS solution containing cell lysate (50 mg / mL) was used to resuspend and act for 10 min, and then centrifuged at 10000 g for 20 minutes, and resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h; before the particles were used, they were resuspended with 7 mL of PBS and then 3 mL of cancer cell lysate component containing adjuvant (protein concentration 50 mg / mL) was added and acted at room temperature for 10 min, to obtain nanoparticles modified by two rounds of cryo-siliconization, addition of cationic substances and anionic substances, which were loaded with lysate inside and outside. The average particle size of the nanoparticles is about 350nm, and the surface potential of the nanovaccine is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 350μg of protein or polypeptide components, and the CpG immune adjuvant used inside and outside each 1mg of PLGA nanoparticles is about 0.02mg in total, with half inside and half outside.

[0131] The steps of the preparation method of the unmodified nanoparticles are basically the same as those of the modified nanoparticles, except that the steps of siliconization, addition of cationic substances and anionic substances are not performed. In the preparation process, the antigen is first loaded inside the nanoparticles by the double emulsion method, and after the antigen (lysate component) is loaded inside, it is centrifuged at 10000g for 20 minutes, and then resuspended with 10mL ultrapure water containing 4% trehalose and freeze-dried for 48h. Before the particles are used, they are resuspended with 7mL PBS and then 3mL cancer tissue lysate components (protein concentration 50mg / mL) containing adjuvants are added and acted at room temperature for 10min to obtain nanoparticles loaded with lysate inside and outside. The average particle size of the nanoparticles is about 320nm, and the surface potential of the nanoparticles is about -5mV; each 1mg PLGA nanoparticle is loaded with about 160μg protein or polypeptide components, and the CpG immune adjuvant used inside and outside each 1mg PLGA nanoparticle is about 0.02mg, and half is inside and half is outside.

[0132] The particle size of the blank nanoparticles is about 300 nm. When preparing the blank nanoparticles, pure water or 8M urea containing an equal amount of CpG is used to replace the corresponding water-soluble components and water-insoluble components.

[0133] (3) Preparation of dendritic cells

[0134] This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare bone marrow-derived dendritic cells (BMDC). First, a 6-8 week old C57 mouse was killed by cervical dislocation, and the tibia and femur of the hind legs were surgically removed and placed in PBS, and the muscle tissue around the bone was removed cleanly with scissors and tweezers. Cut off the two ends of the bone with scissors, and then extract the PBS solution with a syringe, insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly flush the bone marrow into a culture dish. Collect the bone marrow solution, centrifuge at 400g for 3min, and then add 1mL of red blood cell lysis solution to lyse the red blood cells. Add 3mL of RPMI 1640 (10% FBS) culture medium to terminate the lysis, centrifuge at 400g for 3min, and discard the supernatant. The cells were placed in a 10mm culture dish and cultured, using RPMI 1640 (10% FBS) culture medium, and recombinant mouse GM-CSF (20ng / mL) was added at 37 degrees and 5% CO2 for 7 days. On the third day, the culture flask was gently shaken and supplemented with the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL). On the sixth day, half of the medium was replaced. On the seventh day, a small amount of suspended and semi-adherent cells were collected and detected by flow cytometry. + CD80 + When the proportion of cells in CD11c+ cells is between 15-20%, the induced cultured BMDCs can be used for the next experiment.

[0135] (4) Activation of dendritic cells

[0136] Mouse BMDCs were plated on a cell culture plate, and 5 mL of RPMI 1640 (10% FBS) medium was added to every 100,000 DC cells. Then, 20 μg of PLGA nanoparticles loaded with water-soluble components and 20 μg of PLGA nanoparticles loaded with water-insoluble components were added and incubated with BMDCs for 72 h. BMDCs were then collected and centrifuged at 300 g for 5 minutes, washed twice with PBS, and resuspended in PBS for later use.

[0137] (5) Dendritic cell vaccines for cancer prevention

[0138] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. The administration scheme of the dendritic cell vaccine group was as follows: 100 μL of dendritic cell vaccine (1 million dendritic cells) was subcutaneously injected on days 35, 28, 21, 14, and 7 before melanoma inoculation; 1.5×10 5 B16F10 cells. The PBS control group was as follows: 100 μL PBS was injected subcutaneously on the 35th, 28th, 21st, 14th and 7th days before melanoma inoculation; 1.5×105 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse on day 0. Blank nanoparticle or free lysate control group: 100 μL blank nanoparticle or free lysate activated dendritic cells were injected subcutaneously on the 35th, 28th, 21st, 14th and 7th days before melanoma inoculation; 1.5×105 B16F10 cells were subcutaneously inoculated in the lower right back of each mouse on day 0. 5 In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0139] (6) Experimental results

[0140] like Figure 4 As shown, the tumors of mice in the control group grew, while the tumor growth rate of mice immunized with dendritic cell vaccines activated by nanoparticles loaded with antigens was significantly slowed down. Moreover, the dendritic cell vaccine activated by nanoparticles modified by siliconization and adding charged substances has a better preventive effect on melanoma than the nanoparticle-activated dendritic cell vaccine group that was not modified during the preparation process.

[0141] Example 4 Nanoparticles loaded with whole cell components of colon cancer tumor tissue and cancer cells activate dendritic cells in vitro to treat colon cancer

[0142] This example uses mouse colon cancer as a cancer model to illustrate how to use dendritic cell vaccines to treat colon cancer. In this example, MC38 mouse colon cancer cells are used as a cancer model. First, colon cancer tumor tissue and colon cancer cells are lysed to prepare water-soluble components and water-insoluble components. Then, organic polymer material PLGA is used as a nanoparticle skeleton material, BCG (BCG) is used as an immune adjuvant, and a solvent evaporation method is used to prepare nanoparticles, and the nanoparticles are used to activate dendritic cells in vitro, and then the dendritic cells are infused back to treat colon cancer.

[0143] (1) Lysis of tumor tissue and cancer cells and collection of components

[0144] Each C57BL / 6 mouse was subcutaneously inoculated with 2 × 10 6 When the tumors grow to a volume of about 1000 mm 3 The mice were killed and the tumor tissues were removed. The tumor tissues were cut into pieces and ground, and an appropriate amount of pure water was added through a cell filter and repeatedly frozen and thawed 5 times, and ultrasound was used to destroy the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed greater than 5000g for 5 minutes and the supernatant was taken as the water-soluble component soluble in pure water; 8M urea was added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water was converted into a soluble component in 8M urea aqueous solution.

[0145] After the cultured MC38 cancer cell line is collected, it is centrifuged at 350g for 5 minutes, and then the supernatant is discarded and washed twice with PBS, and then the cells are resuspended in ultrapure water and repeatedly frozen and thawed 5 times, and ultrasound can be used to destroy the lysed cells. After the cells are lysed, the lysate is centrifuged at a speed of 3000g for 6 minutes and the supernatant is taken as the water-soluble component soluble in pure water; 8M urea is added to the obtained precipitate to dissolve the precipitate, and the water-insoluble component insoluble in pure water can be converted into a soluble component in 8M urea aqueous solution.

[0146] The water-soluble components from MC38 tumor tissue and MC38 cancer cells and the water-insoluble components dissolved in 8M urea were mixed in a ratio of 1:1 to serve as raw material sources for preparing nanoparticles.

[0147] (2) BCG lysis and collection of components

[0148] The method for lysing BCG and collecting its components are the same as those for lysing cancer cells and collecting its components.

[0149] (3) Preparation of Nanoparticles

[0150] The nanoparticles in this example and the blank nanoparticles used as a control were prepared by the double emulsion method in the solvent evaporation method, and the double emulsion method was appropriately modified and improved. The preparation method was the same as that in Example 1, except that the water-soluble component or the water-insoluble component in Example 1 was replaced by the corresponding mixture in this example.

[0151] (4) Preparation of dendritic cells

[0152] Same as Example 3.

[0153] (5) Activation of dendritic cells

[0154] Same as Example 3.

[0155] (6) Dendritic cell cancer vaccines for cancer treatment

[0156] The control groups of this study were PBS group, blank nanoparticles or free lysate group stimulated BMDC group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. On day 0, 2×10 6 MC38 cells. The dosing regimen for the dendritic cell vaccine group was as follows: 200 μL of vaccine containing 1 million dendritic cells was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after tumor inoculation. The PBS control group regimen was as follows: 200 μL of PBS was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after cancer cell inoculation. Blank nanoparticle or free lysate control group: 200 μL of dendritic cells stimulated with 500,000 blank nanoparticles or free lysate was subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days after tumor inoculation. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0157] (7) Experimental results

[0158] like Figure 5 As shown, the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger, and the tumor growth rate of mice in the vaccine group was significantly slower than that in the control group, and some mice had no tumors and recovered. In summary, the dendritic cell vaccine of the present invention has a good therapeutic effect on colon cancer.

[0159] Example 5 Whole cell components of melanoma tumor tissue and lung cancer tumor tissue loaded on nanoparticles to activate dendritic cells for the treatment of melanoma

[0160] This example uses melanoma as a cancer model to illustrate how to use nanoparticles loaded with whole cell components of melanoma tumor tissue and lung cancer tumor tissue to activate dendritic cells, and use the cell vaccine to treat melanoma. In this example, B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue are first lysed to prepare water-soluble components and water-insoluble components of tumor tissue. Then, organic polymer material PLGA is used as a nanoparticle skeleton material, manganese particles and CpG are used as immune adjuvants, and a solvent volatilization method is used to prepare nanoparticles loaded with tumor tissue components, and then the nanoparticles are used to activate dendritic cells, and the dendritic cell vaccine is used to treat melanoma.

[0161] (1) Lysis of tumor tissue and collection of various components

[0162] Each C57BL / 6 mouse was subcutaneously inoculated with 1.5 × 10 5 B16F10 cells or 2×10 6 LLC lung cancer cells, when the tumor grows to a volume of approximately 1000mm 3 The mice were killed and the tumor tissues were removed. The tumor lysis and component collection methods were the same as in Example 1. The water-soluble components from the B16-F10 tumor tissue and the LLC lung cancer tumor tissue were mixed with the original water-insoluble components dissolved in 8M urea in a ratio of 1:1 to prepare the antigen source of nanoparticles for activating dendritic cells.

[0163] (2) Preparation of antigen-loaded nanoparticles

[0164] Nanoparticle system and the blank nanoparticle in contrast adopt the multiple emulsion method preparation in the solvent evaporation method in the present embodiment, and multiple emulsion method has been carried out suitable modification and improvement, and adopts low temperature siliconization technology and two kinds of modification methods of adding charged substances to improve the antigenic load in the nanoparticle system preparation process.The nanoparticle of the water-soluble component in the load whole cell component and the nanoparticle of the water-insoluble component in the load whole cell component are prepared respectively when preparing, and then use together when using.The nanoparticle preparation material PLGA molecular weight adopted is 24KDa-38KDa, and the immunoadjuvant adopted is manganese colloidal particles and CpG and manganese particles are distributed in the nanoparticle inside and CpG is distributed in the nanoparticle surface.Prepare manganese adjuvant earlier, then adopt the multiple emulsion method to prepare the nanoparticle of internal load antigen and adjuvant after the water-soluble component or the water-insoluble component in the manganese adjuvant and the whole cell component are mixed as the first aqueous phase. When preparing the manganese adjuvant, first add 1 mL of 0.3M Na3PO4 solution to 9 mL of normal saline, then add 2 mL of 0.3M MnCl2 solution, and after standing overnight, Mn2OHPO4 colloidal manganese adjuvant is obtained, and the particle size of the manganese adjuvant is about 13 nM. Then the manganese adjuvant is mixed with the water-soluble component (60 mg / mL) or the water-insoluble component (60 mg / mL) in the whole cell component at a volume ratio of 1:3, and then the antigen and manganese adjuvant are loaded into the nanoparticles by the double emulsion method. After internal loading of antigen (lysate component) and adjuvant, 100 mg of nanoparticles were centrifuged at 10000 g for 20 min, then resuspended in 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (50 mg / mL), centrifuged at 10000 g for 20 min, and then lysed with 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate and 1.0 mM HCl, pH 5.0). 3.0) and fixed at room temperature for 10 min, then fixed at -80°C for 24 h, washed by centrifugation with ultrapure water, resuspended in 3 mL of PBS containing histone (5 mg / mL) and polyarginine (10 mg / mL) for 10 min, then washed by centrifugation at 10000 g for 20 min, resuspended in 10 mL of PBS solution containing cell lysate (50 mg / mL) for 10 min, then centrifuged at 10000 g for 20 min, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h; before incubating the nanoparticles with dendritic cells, they were resuspended in 7 mL of PBS and then 3 mL of cancer tissue lysate component containing CpG adjuvant (protein concentration 50 mg / mL) was added and allowed to react at room temperature for 10 min to obtain nanoparticles modified by cryo-siliconization and addition of cationic substances with lysate loaded both inside and outside.The average particle size of the nanoparticles is about 360nm, and the surface potential of the nanoparticles is about -3mV; each 1mg of PLGA nanoparticles is loaded with about 320μg of protein or polypeptide components, and the CpG adjuvant used inside and outside each 1mg of PLGA nanoparticles is 0.01mg.

[0165] The particle size of the blank nanoparticles is about 300 nm. When preparing the blank nanoparticles, pure water or 8M urea containing equal amounts of manganese adjuvant and CpG adjuvant are used to replace the corresponding water-soluble components and water-insoluble components.

[0166] (4) Preparation of dendritic cells

[0167] Same as Example 1.

[0168] (5) Activation of dendritic cells

[0169] Same as Example 1.

[0170] (6) Dendritic cell cancer vaccines for cancer treatment

[0171] The control groups of this study were PBS group, blank nanoparticles or free lysate group stimulated BMDC group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare melanoma-bearing mice. On day 0, 1.5×10 5 B16F10 cells. The dosing regimen of the dendritic cell vaccine group is as follows: 50 μL of vaccine containing 500,000 dendritic cells is injected intradermally on the 4th, 7th, 10th, 15th and 20th day after melanoma inoculation. The PBS control group is as follows: 50 μL of PBS is injected intradermally on the 4th, 7th, 10th, 15th and 20th day after melanoma inoculation. Blank nanoparticle or free lysate control group: 50 μL of dendritic cells stimulated with blank nanoparticles or free lysate is injected intradermally on the 4th, 7th, 10th, 15th and 20th day after melanoma inoculation. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume is calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0172] (7) Experimental results

[0173] like Figure 6As shown, the tumors of mice in the PBS control group and the blank nanoparticle control group grew larger, and the tumor growth rate of mice in the vaccine group was significantly slower than that of the control group, and some mice had no tumors and recovered. In summary, the dendritic cell vaccine of the present invention has a good therapeutic effect on cancer.

[0174] Example 6 Water-soluble components of colon cancer and lung cancer cells loaded inside and on the surface of microparticles to activate dendritic cells for the treatment of colon cancer

[0175] This example illustrates how to prepare a micron particle system loaded with only the water-soluble parts of colon cancer and lung cancer cell components. In this example, MC38 colon cancer tumor tissue and LLC lung cancer cells were first lysed to prepare water-soluble components and water-insoluble components. Then, organic polymer material PLGA was used as the micron particle skeleton material, manganese particles and poly (I: C) were used as immune adjuvants to prepare micron particles loaded with the water-soluble components of whole cells, and then the particle system was used to activate dendritic cells in vitro and then re-infused to treat colon cancer.

[0176] (1) Lysis of MC38 colon cancer tumor tissue and LLC lung cancer cells and collection of various components

[0177] The collection and lysis methods of tumor tissue and cancer cells and the collection methods of water-soluble components and water-insoluble components are the same as above. The water-soluble components of the two lysates from colon cancer tumor tissue and lung cancer cells obtained above are mixed in a ratio of 3:1 to prepare the antigen source of the micron particle system.

[0178] (2) Preparation of micron particle system

[0179] In the present embodiment, prepare micronized particles and blank micronized particles as a control and adopt the multiple emulsion method in the solvent evaporation method, the micronized particles prepared by the material is that the molecular weight of the organic polymer material PLGA is 38KDa-54KDa, and the immunoadjuvant adopted is manganese particles and poly (I: C) and the manganese particles are distributed in the vaccine and poly (I: C) is distributed on the particle surface. In the preparation process, first prepare manganese adjuvant, then mix the water-soluble components in manganese adjuvant and the whole cell component as the first aqueous phase and adopt the multiple emulsion method to prepare the micronized particles of internal load antigen and adjuvant. When preparing manganese adjuvant, first by the Na3PO4 solution of 0.05mL 0.6M joins in 0.95mL normal saline, then adds the MnCl2 solution of 0.1mL 0.3M, namely obtains Mn2OHPO4 colloidal manganese adjuvant, and the manganese adjuvant particle diameter is about 20nm. Then, the manganese adjuvant was mixed with the water-soluble component (60 mg / mL) in the whole cell component at a volume ratio of 1:4, and the antigen and manganese adjuvant were loaded into the microparticles by the double emulsion method. After the antigen (lysis component) and adjuvant were loaded inside, 100 mg of microparticles were centrifuged at 10000g for 20 minutes, then resuspended with 8 mL of PBS, and then 2 mL of lysis solution component containing poly (I: C) adjuvant (protein concentration 80 mg / mL) was added and reacted at room temperature for 10 minutes to obtain a microparticle system. The average particle size of the microparticles is about 1.5 μm, and the surface potential of the microparticles is about -4 mV; each 1 mg of PLGA microparticles is loaded with about 200 μg of protein or polypeptide components, and the poly (I: C) adjuvant used for each 1 mg of PLGA microparticles is 0.01 mg.

[0180] The particle size of the blank micronized particles is about 1.4 μm. When preparing the blank micronized particles, pure water containing equal amounts of manganese adjuvant and poly(I:C) adjuvant is used to replace the corresponding water-soluble components.

[0181] (4) Preparation of dendritic cells

[0182] Same as Example 1.

[0183] (5) Activation of dendritic cells

[0184] Same as Example 1, 20 ng / mL GM-CSF and 20 ng / mL IL-2 were added to the cell culture medium during the co-incubation process.

[0185] (6) Dendritic cell vaccines for cancer treatment

[0186] The control groups of this study were PBS group, blank microparticles or free lysate group. Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare colon cancer tumor-bearing mice. On day 0, 2×10 6MC38 cells. The dosing regimen for the dendritic cell vaccine group is as follows: 100 μL of 1 million dendritic cells were subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days. The PBS control group regimen is as follows: 100 μL of PBS was subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days. Blank microparticle or cell lysate control group: 100 μL of blank microparticles or free cell lysate in vitro stimulated dendritic cells (1 million dendritic cells) were subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0187] (4) Experimental results

[0188] like Figure 7 As shown, the tumor growth rate of mice in the vaccine administration group was significantly slowed down compared with the control group. Dendritic cell vaccines activated in vitro by microparticles loaded with water-soluble components have a therapeutic effect on colon cancer.

[0189] Example 7 6M guanidine hydrochloride dissolves breast cancer cells and loads them into microparticles to activate dendritic cells in vitro for the prevention of breast cancer

[0190] This example uses 4T1 mouse triple-negative breast cancer as a cancer model to illustrate how to use 6M urea to dissolve whole cell components and prepare a micron particle system loaded with whole cell components, and use the micron particle system to activate dendritic cells in vitro to prevent breast cancer. In this example, breast cancer cells are first inactivated and denatured, and the whole cell components are dissolved after lysing cancer cells with 6M guanidine hydrochloride. Then, PLGA is used as the micron particle skeleton material, CpG and Poly (I: C) are used as immune adjuvants, and a solvent evaporation method is used to prepare a micron particle system loaded with whole cell components, and the micron particle system is used to activate dendritic cells in vitro to prevent breast cancer.

[0191] (1) Lysis of cancer cells and collection of components

[0192] The cultured 4T1 cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cancer cells were inactivated and denatured by ultraviolet light and high temperature heating, respectively, and then the breast cancer cells were lysed with an appropriate amount of 6M guanidine hydrochloride and the lysate was dissolved to serve as the raw material source for preparing the particle system.

[0193] (2) Preparation of micron particle system

[0194] In this embodiment, the preparation of the micron particle system and the blank micron particles as a control adopt the double emulsion method in the solvent evaporation method. The micron particle preparation material used is the organic polymer material PLGA with a molecular weight of 38KDa-54KDa, and the immune adjuvants used are CpG and Poly (I: C). The double emulsion method was appropriately modified and improved during the preparation. In the micron particle preparation process, the double emulsion method was first used to prepare nanoparticles with internal antigen and adjuvant loading. After the internal antigen and adjuvant were loaded, 100mg of micron particles were centrifuged at 10000g for 20 minutes, resuspended with 10mL of ultrapure water containing 4% trehalose and dried for 48h; before the micron particle system was injected, it was resuspended with 8mL PBS and then 2mL of cancer cell lysate components (protein concentration 80mg / mL) were added and acted at room temperature for 10min to obtain the desired micron particle system. The average particle size of the micron particle system is about 1.5μm, and the surface potential of the micron particle system is about -4mV; each 1mg PLGA micron particle is loaded with about 140μg of protein or polypeptide components. The particle size of the blank microparticles is about 1.4 μm. When preparing the blank microparticles, 6 M guanidine hydrochloride containing equal amounts of CpG and Poly (I: C) adjuvants is used to replace the corresponding cell components.

[0195] (3) Preparation of dendritic cells

[0196] Same as Example 1.

[0197] (4) Activation of dendritic cells

[0198] Same as Example 1.

[0199] (5) Micron particle system for cancer prevention

[0200] Female BALB / c mice aged 6-8 weeks were selected to prepare 4T1 tumor-bearing mice. The vaccine prevention group was subcutaneously injected with 100 μL of dendritic cell vaccine (1 million dendritic cells) on days 35, 28, 21, 14, and 7 before tumor inoculation; on day 0, each mouse was subcutaneously inoculated with 4×10 5 The PBS blank control group was subcutaneously injected with 100 μL PBS on days 35, 28, 21, 14, and 7 before tumor inoculation; 4×10 5 4T1 cells. The blank microparticle or cell lysate control group was subcutaneously injected with dendritic cells (1 million) stimulated with lysate or PLGA blank microparticles on days 35, 28, 21, 14, and 7 before tumor inoculation; 4×10 54T1 cells. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculate, where v is the tumor volume, a is the tumor length, and b is the tumor width. In the survival experiment, the tumor volume of mice exceeded 2000mm 3 The mice were considered dead and euthanized.

[0201] (6) Experimental results

[0202] like Figure 8 As shown, compared with the control group, the tumor growth rate in the vaccine prevention group was significantly slowed down and the survival period of mice was significantly prolonged. It can be seen that the vaccine of the present invention has a preventive effect on breast cancer.

[0203] Example 8 Nanoparticle system loaded with tumor tissue and whole cell components of cancer cells activates dendritic cells for the prevention of cancer metastasis

[0204] This example uses a mouse melanoma mouse lung metastasis cancer model to illustrate the use of dendritic cell vaccines to prevent cancer metastasis. In actual application, the specific dosage form, adjuvant, administration time, number of administrations, and administration regimen can be adjusted according to the situation. In this example, mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell lysis component were loaded on the nanoparticle system at a mass ratio of 1:4, and the particle system was used to activate dendritic cells to prevent cancer metastasis. In this example, nanoparticles loaded with four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used as control nanoparticles to analyze the effects of nanoparticles loaded with whole cell antigens and nanoparticles loaded with multiple polypeptide neoantigens in the preparation of dendritic cell vaccines.

[0205] (1) Lysis of tumor tissue and cancer cells

[0206] After collecting mouse B16F10 melanoma tumor tissues and cultured cancer cells, 8M urea was used to lyse and dissolve the whole cell components of the tumor tissues and cancer cells, and then the tumor tissue components and cancer cell components were mixed at a mass ratio of 1:4.

[0207] (2) Preparation of nanoparticle systems

[0208] In the present embodiment, the nanoparticle system and the blank nanoparticles used as the control are prepared by the emulsion method in the solvent evaporation method. The molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa, and the immunoadjuvant used is CpG and CaCl2, and the adjuvant is loaded inside the nanoparticle. The preparation method is as mentioned above. In the preparation process, the emulsion method is first used to load antigen inside the nanoparticle. After the internal load antigen (cracking component), 100mg nanoparticles are centrifuged at 10000g for 20 minutes, and 10mL ultrapure water containing 4% trehalose is resuspended and freeze-dried for 48h for standby use. The average particle size of the nanoparticles is about 320nm; about 160μg protein or polypeptide components are loaded per 1mg PLGA nanoparticles. The preparation method of the control nanoparticles loaded with multiple antigen polypeptides is the same as above, and the control nanoparticle particle size is about 310nm, and about 150μg antigen polypeptides are loaded per 1mg PLGA nanoparticles.

[0209] (3) Preparation of dendritic cells

[0210] Same as Example 1.

[0211] (4) Activation of dendritic cells

[0212] Same as Example 1.

[0213] (5) Dendritic cell vaccines for preventing cancer metastasis

[0214] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. The vaccine group was subcutaneously injected with 100 μL of dendritic cells (1 million) on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The PBS blank control group was subcutaneously injected with 100 μL of PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The control vaccine group was subcutaneously injected with 100 μL of dendritic cells activated by control nanoparticles loaded with polypeptide antigens on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. On day 0, each mouse was injected with 3×10 5 The mice were killed on day 15 and their lungs were removed to observe the number of cancer masses formed by melanoma metastasis in the lungs.

[0215] (6) Experimental results

[0216] like Fig. 9As shown, the number of cancer metastases in the vaccine group mice was significantly reduced compared with the control group. Moreover, the dendritic cell vaccine activated by nanoparticles loaded with whole cell components was more effective than the dendritic cell vaccine activated by nanoparticles loaded with several antigenic polypeptides. This shows that the dendritic cell vaccine activated in vitro by nanoparticles loaded with whole cell components described in the present invention can effectively prevent cancer metastasis.

[0217] Example 9: Pancreatic cancer tumor tissue and colon cancer tumor tissue lysate components loaded inside and on the surface of nanoparticles for the treatment of pancreatic cancer

[0218] This example uses mouse pancreatic cancer as a cancer model to illustrate the use of dendritic cell vaccines to treat cancer. In this example, mouse Pan02 pancreatic cancer tumor tissue and MC38 colon cancer tumor tissue lysate components were loaded onto nanoparticles at a ratio of 2:1.

[0219] First, pancreatic cancer and colon cancer tumor tissues of mice were obtained and lysed to prepare water-soluble components and original water-insoluble components dissolved in 6M guanidine hydrochloride. When preparing particles, the water-soluble component is a mixture of 2:1 of water-soluble components of pancreatic cancer tumor tissue and water-soluble components of colon cancer tumor tissue; the water-insoluble component is a mixture of 2:1 of water-insoluble components of pancreatic cancer tumor tissue and water-insoluble components of colon cancer tumor tissue. PLGA was used as the nanoparticle skeleton material, and nanoparticles were prepared without adding any adjuvants, and the nanoparticles were used to activate dendritic cells and then infused back into the body to treat tumors in Pan02 pancreatic cancer-bearing mice.

[0220] (1) Lysis of tumor tissue and collection of various components

[0221] Each C57BL / 6 mouse was inoculated subcutaneously in the axilla with 2 × 10 6 MC38 colon cancer cells or 1×10 6 Pan02 pancreatic cancer cells were inoculated in each mouse and grew to a volume of approximately 1000 mm 3 The mice were killed and the tumor tissues were removed. The lysis method and the collection method of each component were the same as in Example 1, except that 6M guanidine hydrochloride was used instead of 8M urea to dissolve the water-insoluble components.

[0222] (2) Preparation of Nanoparticles

[0223] The method for preparing nanoparticles in this example is the same as that in Example 1 except that no adjuvant is used.

[0224] (3) Preparation of dendritic cells

[0225] Same as Example 1.

[0226] (4) Activation of dendritic cells

[0227] Same as Example 1, but 20 ng / mL GM-CSF was added to the cell culture medium during the incubation.

[0228] (5) Vaccines for cancer treatment

[0229] Female C57BL / 6 mice aged 6-8 weeks were selected to prepare pancreatic cancer mice. On day 0, 1×10 6 Pan02 cells. The dosing regimen for the dendritic cell vaccine group was as follows: 100 μL of 1 million dendritic cells were subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days, respectively. The PBS control group regimen was as follows: 100 μL of PBS was subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days, respectively. Blank nanoparticle or cell lysate control group: 100 μL of blank nanoparticles or free cell lysate in vitro stimulated dendritic cells (1 million dendritic cells) were subcutaneously injected on the 3rd, 6th, 9th, 12th, 15th and 18th days, respectively. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0230] (4) Experimental results

[0231] like Fig.10 As shown, compared with the control group, the tumor growth rate in the vaccine group was significantly slowed down and the survival time of mice was significantly prolonged. This shows that nanoparticles loaded with cellular components of cancer tumor tissue without adding adjuvants can activate dendritic cells in vitro and use the dendritic cells as a vaccine to treat pancreatic cancer.

[0232] Example 10 Whole cell components of cancer cells loaded on mannose-targeted nanoparticles to activate dendritic cells for cancer prevention

[0233] This example uses a mouse melanoma model to illustrate how to use a dendritic cell vaccine to prevent cancer. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation. In this example, mouse melanoma cancer cells were lysed and dissolved with 8M urea, and then the cancer cell lysis components were loaded on the nanoparticle system. The nanoparticle system can be taken up into the dendritic cells through the mannose receptors on the surface of the dendritic cells.

[0234] (1) Lysis of cancer cells

[0235] After the cultured cancer cells were collected, 8M urea was used to lyse and dissolve the whole cell fraction of the cancer cells.

[0236] (2) Preparation of nanoparticle systems

[0237] Nanoparticle system and nanoparticles with only loaded cell components and no loaded adjuvant as contrast in the present embodiment are prepared by the multiple emulsion method in the solvent evaporation method. The nanoparticles prepared by the material are PLGA and mannose-modified PLGA, and the ratio is 4:1, and the molecular weight is 7KDa-17KDa, and the immunoadjuvants adopted are CpG and adjuvants are loaded inside the nanoparticles. The preparation method is as previously mentioned, and in the preparation process, the multiple emulsion method is first adopted to load cell components inside the nanoparticles, and after the internal loaded cell components, 100mg nanoparticles are centrifuged at 10000g for 20 minutes, and the ultrapure water resuspended after lyophilization of 10mL containing 4% trehalose is used for standby use after 48h. The mean particle diameter of the nanoparticles with target head (loaded adjuvant) and without target head (loaded adjuvant) is about 320nm, and every 1mg PLGA nanoparticles is about loaded with 60μg protein or polypeptide components. The particle size of the control nanoparticles without adjuvant but with target head is also about 320 nm. The same amount of cell components but without any immune adjuvant are used in the preparation. Each 1 mg of PLGA nanoparticles is loaded with about 60 μg of protein or polypeptide components.

[0238] (3) Preparation of dendritic cells

[0239] Same as Example 1.

[0240] (4) Activation of dendritic cells

[0241] Same as Example 1.

[0242] (3) Dendritic cell vaccines for cancer prevention

[0243] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. The vaccine group was subcutaneously injected with 100 μL of dendritic cell vaccine (1 million) on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The PBS blank control group was subcutaneously injected with 100 μL of PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. Each mouse was subcutaneously injected with 1.5×10 5 B16F10 melanoma cells. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3The mice were considered dead and euthanized.

[0244] (4) Experimental results

[0245] As shown in Figure 11, the tumor growth rate of mice in the vaccine group was significantly slower than that of the control group. Moreover, the dendritic cell vaccine activated by nanoparticles containing target heads was more effective than the dendritic cell vaccine activated by nanoparticles without target heads, and the dendritic cell vaccine activated by nanoparticles containing immune adjuvants was better than the dendritic cell vaccine activated by nanoparticles without immune adjuvants. This shows that the vaccine of the present invention can prevent cancer, and the addition of target heads and adjuvants helps the nanoparticles to activate dendritic cell vaccines in vitro to play a role.

[0246] Example 11 Whole cell components of cancer cells loaded on nanoparticles with BCG as adjuvant to activate dendritic cells in vitro for the prevention of liver cancer

[0247] This example uses BCG as an immune adjuvant to illustrate how to prepare nanoparticles loaded with whole cell components of liver cancer cells and use the particles to activate dendritic cells in vitro to prevent liver cancer. In this example, PLGA is first used as a nanoparticle skeleton material, BCG is used as an immune adjuvant, and a nanoparticle system is prepared by a solvent evaporation method, and then the particle system is used to activate dendritic cells and used to prevent liver cancer.

[0248] (1) Lysis of cancer cells and collection of components

[0249] In this example, cancer cell lysis and lysate collection are the same as above.

[0250] (2) Cancer cell lysis and collection of components

[0251] The method for lysing cancer cells and collecting and solubilizing the lysate in this example is the same as the lysing method in Example 1, except that the tumor tissue is replaced with cancer cells.

[0252] (3) Preparation of nanoparticle systems

[0253] The preparation method of the nanoparticles and the materials used in this example are the same as those in Example 1. However, in this example, the immune adjuvant loaded by the nanoparticles is replaced by BCG instead of poly(I:C).

[0254] (3) Preparation of dendritic cells

[0255] Same as Example 1.

[0256] (4) Activation of dendritic cells

[0257] Same as Example 1.

[0258] (5) Dendritic cell vaccines for cancer prevention

[0259] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. The vaccine group was subcutaneously injected with 100 μL of dendritic cell vaccine (1 million) on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The PBS blank control group was subcutaneously injected with 100 μL of PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The lysate control group was subcutaneously injected with 100 μL of dendritic cells stimulated by free lysate (1 million) on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. Each mouse was subcutaneously injected with 2×10 6 Hepa1-6 liver cancer cells. In the experiment, the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b 2 Calculated as follows: where v is the tumor volume, a is the tumor length, and b is the tumor width. For animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeds 2000mm 3 The mice were considered dead and euthanized.

[0260] (6) Experimental results

[0261] like Fig.12 As shown, compared with the control group, the tumor growth rate of the vaccine-administered group was significantly slowed down and the survival period of mice was significantly prolonged. It can be seen that the vaccine of the present invention can prevent liver cancer.

[0262] Example 12 Nanoparticle system loaded with whole cell components of tumor tissue and cancer cells activates dendritic cells for cancer prevention

[0263] This example uses a mouse melanoma mouse model to illustrate the use of dendritic cell vaccines to prevent cancer. In actual application, the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation. In this example, mouse melanoma tumor tissue and cancer cells were lysed and dissolved with 8M urea, and then the tumor tissue lysis component and the cancer cell lysis component were loaded on the nanoparticle system at a mass ratio of 1:1, and the particle system was used to activate dendritic cells to prevent cancer. In this example, nanoparticles loaded with four polypeptide neoantigens B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188 (SVYDFFVWL) were used as control nanoparticles to analyze the effects of nanoparticles loaded with whole cell antigens and nanoparticles loaded with multiple polypeptide neoantigens in the preparation of dendritic cell vaccines. In this example, the biocalcified nanoparticles were loaded with whole cell antigens inside and on the surface of the nanoparticles and then incubated with dendritic cells.

[0264] (1) Lysis of tumor tissue and cancer cells

[0265] After collecting mouse B16F10 melanoma tumor tissues and cultured cancer cells, 8M urea was used to lyse and dissolve the whole cell components of the tumor tissues and cancer cells, and then the tumor tissue components and cancer cell components were mixed at a mass ratio of 1:4.

[0266] (2) Preparation of nanoparticle systems

[0267] In the present embodiment, the nanoparticle system and the blank nanoparticles used as the control are prepared by the emulsion method in the solvent evaporation method. The nanoparticle preparation material PLGA molecular weight used is 7KDa-17KDa, and the immunoadjuvant CpG and Poly (I: C) used are loaded inside the nanoparticle. The preparation method is as follows, in the preparation process, the emulsion method is first used to load antigen inside the nanoparticle, after the internal load antigen (cracking component), 100mg PLGA nanoparticles are resuspended using 18mL PBS, then 2mL is added to be dissolved in the tumor tissue and cancer cell lysate (60mg / mL) of 8M urea, and the precipitation is collected after 10000g centrifugation for 20 minutes after 10 minutes of room temperature action. Then the 100mg PLGA nanoparticles are resuspended in 20mL DMEM culture medium, then 200μL of CaCl2 (1mM) is added and reacted for two hours at 37°C. Then the precipitation is collected after 10000g centrifugation for 20 minutes, and the ultrapure water is resuspended and centrifuged and washed twice. Then, the 100 mg nanoparticles were resuspended in 10 mL RPMI 1640 medium and incubated with dendritic cells. The average particle size of the nanoparticles was about 320 nm; each 1 mg PLGA nanoparticle was loaded with about 150 μg of protein or polypeptide components. The preparation method of the control nanoparticles loaded with multiple antigen polypeptides was the same as above, the particle size of the control nanoparticles was about 310 nm, and each 1 mg PLGA nanoparticle was loaded with about 150 μg of antigen polypeptides.

[0268] (3) Preparation of dendritic cells

[0269] Same as Example 1.

[0270] (4) Activation of dendritic cells

[0271] Same as Example 1.

[0272] (5) Dendritic cell vaccines for cancer prevention

[0273] Female C57BL / 6 mice aged 6-8 weeks were selected as model mice to prepare tumor-bearing mice. The vaccine group was subcutaneously injected with 100 μL of dendritic cells (1 million) on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The PBS blank control group was subcutaneously injected with 100 μL of PBS on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. The control vaccine group was subcutaneously injected with 100 μL of dendritic cells activated by control nanoparticles loaded with polypeptide antigens on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation. Each mouse was subcutaneously injected with 1.5×10 5B16F10 melanoma cells were added, and then the size of the mouse tumor volume was recorded every 3 days starting from the 3rd day. The tumor volume was calculated using the formula v = 0.52 × a × b2, where v is the tumor volume, a is the tumor length, and b is the tumor width. For the sake of animal experiment ethics, in the mouse survival test, when the mouse tumor volume exceeded 2000mm3, the mouse was considered dead and euthanized.

[0274] (6) Experimental results

[0275] like Fig.13 As shown, compared with the control group, the growth rate of cancer tumors in the vaccine group mice was significantly slowed down and the tumors disappeared in some mice after inoculation of cancer cells. Moreover, the dendritic cell vaccine activated by nanoparticles loaded with whole cell components was more effective than the dendritic cell vaccine activated by nanoparticles loaded with several antigenic peptides. This shows that the dendritic cell vaccine activated in vitro by nanoparticles loaded with whole cell components described in the present invention can effectively prevent cancer.

[0276] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Use of a dendritic cell vaccine in the preparation of a drug for treating or preventing cancer, characterized in that: The dendritic cell vaccine is obtained by activating dendritic cells in vitro by delivery particles loaded with cell components solubilized by a dissolving agent; wherein, The delivery particles are nanoparticles and / or micron particles, and the preparation materials of the delivery particles are organic synthetic polymer materials, natural polymer materials or inorganic materials; The cell components solubilized by the lytic agent are lysed by a solubilizing solution containing a solubilizing agent to lyse cancer cells and / or tumor tissues, and then the lysate components are dissolved by a solubilizing solution containing a solubilizing agent; the solubilizing agent is selected from urea and / or guanidine salt; In the delivery particles loaded with the cell components solubilized by the solubilizing agent, 1 mg of the delivery particle preparation material carries 0.06 mg - 0.35 mg of the protein / peptide component; The in vitro activation is to co-incubate the delivery particles of the cell components solubilized by the loading dissolving agent with the dendritic cells. During the in vitro activation, the ratio of the dendritic cells to the delivery particles of the cell components solubilized by the loading dissolving agent is 1 million dendritic cells corresponding to 0.4 mg - 0.6 mg delivery particles; The cancer is melanoma, colon cancer, breast cancer, pancreatic cancer or liver cancer, When the cancer is melanoma, the cancer cells and / or tumor tissue are cancer cells and / or tumor tissue of melanoma; when the cancer is colon cancer, the cancer cells and / or tumor tissue are cancer cells and / or tumor tissue of colon cancer; when the cancer is breast cancer, the cancer cells and / or tumor tissue are cancer cells and / or tumor tissue of breast cancer; when the cancer is pancreatic cancer, the cancer cells and / or tumor tissue are cancer cells and / or tumor tissue of pancreatic cancer; when the cancer is liver cancer, the cancer cells and / or tumor tissue are cancer cells and / or tumor tissue of liver cancer.

2. The use according to claim 1, characterized in that: The dendritic cells are autologous dendritic cells and / or allogeneic dendritic cells.

3. The use according to claim 1, characterized in that: The cell components solubilized by the lytic agent are loaded onto the surface of the delivery particles in a manner that includes at least one of adsorption, covalent bonding, charge interaction, hydrophobic interaction, one-step or multiple-step solidification, mineralization and encapsulation.

4. The use according to claim 1, characterized in that: The surface of the delivery particle is loaded with one or more layers of cell components solubilized by a dissolving agent, wherein when multiple layers of cell components solubilized by a dissolving agent are loaded, there are modifications between the layers.

5. The use according to claim 1, characterized in that: The interior and / or surface of the delivery particles are loaded with an immune enhancing adjuvant.

6. The use according to claim 1, characterized in that: The surface of the delivery particle is connected with a target head that actively targets dendritic cells.

7. The use according to claim 1, characterized in that: The cell components solubilized by the lytic agent are loaded inside the delivery particles, and / or the cell components solubilized by the lytic agent are loaded on the surface of the delivery particles.

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