Preparation method and application of an autoimmune disease vaccine derived from pre-activated antigen-presenting cells

Abstract

The present invention relates to a method for preparing an autoimmune disease vaccine derived from pre-activated antigen-presenting cells and its application. The preparation method comprises the following steps: co-incubating antigen-presenting cells with a first delivery particle loaded with whole-cell antigens of autoimmune diseases to obtain pre-activated antigen-presenting cells; preparing nanovesicles from the cell membranes of the pre-activated antigen-presenting cells or nanoparticles or microparticles with cell membrane components loaded on the surface thereof to obtain an autoimmune disease vaccine. While the present invention realizes the loading of a broad-spectrum and diverse range of cancer cell antigens by dendritic cell-derived vaccines, it overcomes the problems of live cell vaccines such as the difficulty in maintaining the activity of dendritic cells and the inability to be freeze-dried for long-term storage, and can prepare vaccines loaded with broad-spectrum autoimmune disease antigen epitopes for the prevention and treatment of autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and particularly to a preparation method and application of an autoimmune disease vaccine derived from pre-activated antigen-presenting cells. Background Art

[0002] Autoimmune diseases are a class of diseases that seriously threaten human health. Their pathogenesis is that the human immune system, for some reason, misidentifies its own substances as foreign antigens and launches an attack on cells or tissues containing such antigens, resulting in a series of consequences and diseases. Taking type I diabetes as an example, for some reasons that have not been fully studied, the body's immune system misidentifies some components in the islets or β cells as foreign antigens, thus initiating an attack on β cells and then killing most of the β cells. Since β cells are the substances in the human body that secrete insulin, and insulin is a key substance for controlling blood sugar, when most of the β cells are killed, the human body cannot produce enough insulin to control blood sugar, so type I diabetes occurs. Therefore, if the attack of the human immune system on β cells can be prevented or relieved, type I diabetes can be prevented or reversely treated. And a vaccine that can induce immune tolerance in the body is one of the main methods to achieve the above purpose. Since β cells are mainly attacked by effector T cells (T eff ), it is crucial to induce regulatory T cells (T eff ) that can recognize the same antigen as effector T cells but have an inhibitory effect on effector T cells (T reg ). Therefore, the present invention aims to find a method for efficiently inducing the generation of regulatory T cells, so as to find a method for effectively preventing and treating autoimmune diseases. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a nano-vaccine (Nanovaccine, NP) or micro-vaccine (Microvaccine, MP) derived from antigen-presenting cells activated by nanoparticles and / or microparticles, and prepared with the membrane components of the antigen-presenting cells, which can be used for the prevention or treatment of autoimmune diseases.

[0004] The present invention provides a preparation method of an autoimmune disease vaccine derived from pre-activated antigen-presenting cells, comprising the following steps:

[0005] S1. Co-incubate antigen-presenting cells with a first delivery particle loaded with whole cell antigens of autoimmune diseases to obtain pre-activated antigen-presenting cells;

[0006] S2. Prepare the cell membrane of the pre-activated antigen-presenting cells into nanovesicles to obtain an autoimmune disease vaccine;

[0007] Alternatively, the cell membrane components of pre-activated antigen-presenting cells are loaded onto the second delivery particles loaded with the whole cell antigen of autoimmune diseases to obtain an autoimmune disease vaccine;

[0008] Wherein,

[0009] The first delivery particle or the second delivery particle is independently a nanoparticle or a microparticle;

[0010] The whole cell antigen of autoimmune diseases is prepared by the following steps: freezing cells or tissues containing autoimmune disease antigens, adding water for freeze-thaw lysis, collecting the supernatant and the soluble part in the precipitate after being dissolved by a solubilizer to obtain the whole cell antigen of autoimmune diseases; or lysing cells or tissues containing autoimmune disease antigens with a solubilizer and collecting the soluble part to obtain the whole cell antigen of autoimmune diseases.

[0011] Furthermore, the solubilizer is selected from one or more of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, proteolytic enzymes, albumin, lecithin, inorganic salts, Triton, Tween, amino acids, glycosides, and choline.

[0012] Furthermore, while loading the cell membrane components of pre-activated antigen-presenting cells onto the second delivery particles loaded with the whole cell antigen of autoimmune diseases, it also includes loading the cell membrane components of cells containing autoimmune disease antigens onto the second delivery particles. Wherein, the cell membrane components include cell membranes and / or extracellular vesicle membranes.

[0013] Furthermore, when the delivery particles (hereinafter, "delivery particles" all refer to the first delivery particle or the second delivery particle) simultaneously load the membrane components of antigen-presenting cells and the membrane components of cells containing autoimmune disease antigens (hereinafter, the membrane components of β cells are taken as an example for illustration), the specific steps include:

[0014] (1) Co-incubating one or more antigen-presenting cells with nanoparticles and / or microparticles loaded with the whole cell antigen of autoimmune diseases for a certain period of time to activate the antigen-presenting cells;

[0015] (2) Mechanically disrupting, membrane filtering, gradient centrifuging or chemically treating the activated antigen-presenting cells to obtain cell membrane fragments and / or nanovesicles derived from the antigen-presenting cells;

[0016] (3) Mechanically disrupting, membrane filtering, gradient centrifuging or chemically treating cells containing autoimmune disease antigens (such as β cells) to obtain cell membrane fragments and / or nanovesicles derived from β cells;

[0017] (4) Co-acting the products obtained in steps (2) and (3) with the second delivery particles loaded with the whole cell antigen of autoimmune diseases to obtain the desired product.

[0018] Further, the mechanical disruption method is selected from one or more of ultrasound, homogenization, homogenate, high-speed stirring, high-pressure disruption, high-shear force disruption, swelling, and shrinkage.

[0019] Further, the co-action method is selected from one or more of co-incubation, ultrasound, co-extrusion, ultrafiltration, dialysis, stirring, homogenization, and homogenate.

[0020] Further, in step S1, during co-incubation, the incubation system contains cytokines and / or antibodies; the cytokines are selected from one or more of interleukin 1 receptor antagonist, growth factor, interleukin, interferon, tumor necrosis factor, colony-stimulating factor, activin, and inhibin; the antibodies are selected from one or more of PD1 antibody, PD-L1 antibody, CTAL-4 antibody, TIGIT antibody, TIM-3 antibody, LAG-3 antibody, αCD-8 antibody, αCD-28 antibody, αCD-40 antibody, αOX-40 antibody, and αOX-40L antibody.

[0021] Further, the cytokines include but are not limited to interleukin 1 receptor antagonist (IL-1ra), interleukin 2 (IL-2), transforming growth factor-β (TGF-β), interleukin 7 (IL-7), interleukin 10 (IL-10), interleukin 14 (IL-14), interleukin 4 (IL-4), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 21 (IL-21), interleukin 17 (IL-17), interleukin 12 (IL-12), interleukin 6 (IL-6), interleukin 33 (IL-33), interferon-γ (IFN-γ), TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), activins, and inhibins.

[0022] Preferably, the incubation system includes one of the following combinations:

[0023] (1) GM-CSF, IL-2, IL-7, IL-10, and CD40 antibody;

[0024] (2) TGF-β, IL-4, IL-7, IL-10 (most preferably);

[0025] (3) GM-CSF, IL-2, IL-4, IL-10, and PD-L1 antibody (the PD-L1 antibody is an optional substance);

[0026] (4) TGF-β, IL-10, IL-4, and PD-L1 antibody;

[0027] (5) GM-CSF, IL-2, IL-10 and TGF-β;

[0028] (6) IL-2, IL-10, IL-4 and TGF-β;

[0029] (7) IL-10, IL-2, IL-4 and IL-13;

[0030] (8) IL-13, IL-10, IL-4 and TGF-β;

[0031] (9) GM-CSF, IL-2, IL-10 and IL-13.

[0032] Furthermore, in step S1, glucose and / or thapsigargin are contained in the co-incubation system during co-incubation.

[0033] Furthermore, the antigen-presenting cell (APC) is selected from at least one of dendritic cells (DC), B cells, and macrophages.

[0034] The autoimmune diseases described in the present invention include but are not limited to type I diabetes, rheumatoid arthritis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, ulcerative colitis, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, and thyroid autoimmune disease.

[0035] The nano-vaccine or micro-vaccine described in the present invention is characterized in that: when the autoimmune disease is type I diabetes, the cell containing the antigen related to type I diabetes is a β cell; the tissue is a tissue containing β cells, such as pancreatic tissue and / or islet tissue.

[0036] Furthermore, the activated antigen-presenting cell can be appropriately washed before preparing the nano-vaccine, and the washing solution used in the washing process contains a protease inhibitor.

[0037] Furthermore, the process of ultrasonic treatment of the antigen-presenting cell for preparing the nano-vaccine or micro-vaccine is low-power ultrasound (below 500 W); the gradient centrifugation for preparing the nano-vaccine or micro-vaccine is gradient centrifugation with sequentially increasing centrifugation speed; the pore size of the filter membrane used for membrane filtration in preparing the nano-vaccine or micro-vaccine decreases sequentially from large to small; the co-action time is greater than 20 seconds.

[0038] Furthermore, the particle size of the nano-vaccine is greater than 30 nm and less than 1000 nm; the particle size of the micro-vaccine is greater than 1 μm and less than 50 μm.

[0039] Furthermore, the particle size range of the nanoparticles for activating antigen-presenting cells is from 10 nm to 1000 nm, and the particle size range of the microparticles for activating antigen-presenting cells is from 1 μm to 50 μm.

[0040] Furthermore, the interior and / or surface of the first delivery particle or the second delivery particle is loaded with immune-suppressing substances.

[0041] Furthermore, the immune-suppressing substances are selected from one or more of immune inhibitors of microbial origin, products of the human or animal immune system, mRNA, DNA, innate immune inhibitors, adaptive immune inhibitors, chemically synthesized drugs, fungal polysaccharides, and traditional Chinese medicines; they can also be selected from one or more of glucocorticoid drugs, calcineurin inhibitors, antimetabolites, antibodies, cytokines, alkylating agents, plant medicine components, and mineral medicine components; they can also be selected from one or more of cyclosporine, rapamycin, tacrolimus, gusperimus, fingolimod, methylprednisolone, tripterygium wilfordii, mycophenolate mofetil, cyclophosphamide, azathioprine, everolimus, sandimmun, sangcya, cyclosporine polypeptide A, cyclosporine, newchudiming, anti-IL-2 receptor monoclonal antibody, TGF-β, interleukin, ginseng, and astragalus membranaceus.

[0042] Furthermore, the first delivery particle or the second delivery particle is loaded with one or more of positively charged polypeptides (such as KALA polypeptide, RALA polypeptide, melittin, etc.), arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH4HCO3, protamine, and histone.

[0043] Furthermore, the surface of the nano-vaccine or the micro-vaccine is connected with a targeting head with an active targeting function, and the targeting head can be mannose, mannan, CD19 antibody, CD20 antibody, BCMA antibody, CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, etc.

[0044] The present invention also provides an application of the above-mentioned autoimmune disease vaccine in the preparation of a drug for treating or preventing autoimmune diseases.

[0045] Furthermore, the antigen-presenting cells are derived from one or more of autologous, allogeneic, cell lines, or stem cell differentiation.

[0046] Furthermore, the ways of loading the whole cell antigen on the surface of the nanoparticles or microparticles include at least one of adsorption, covalent connection, charge interaction, hydrophobic interaction, one-step or multi-step solidification, mineralization, and encapsulation.

[0047] Furthermore, the nanoparticles or microparticles are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials, and can be prepared by existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, microfluidic methods, extrusion methods, hot melt methods.

[0048] Furthermore, the organic synthetic polymer materials include but are not limited to poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol (PEG), polycaprolactone (PCL), Poloxamer, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethyleneimine (PEI), polytrimethylene carbonate (PTMC), polyanhydrides, PDON, PPDO, polymethyl methacrylate (PMMA), polyamino acids, synthetic polypeptides, etc.; the natural polymer materials include lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, polypeptides, etc.; the inorganic materials include iron(III) oxide, iron(II,III) oxide, carbonates, phosphates, etc.

[0049] Furthermore, the nanoparticles or microparticles may not be modified during the preparation process, or appropriate modification techniques may be used to increase the antigen loading amount of the nanoparticles or microparticles. The modification techniques include but are not limited to biomineralization (such as silicification, calcification, magnesiation), gelation, crosslinking, chemical modification, addition of charged substances, etc.

[0050] Furthermore, the ways of loading antigens onto the surface of nanoparticles or microparticles include but are not limited to adsorption, covalent connection, charge interaction (such as adding positively charged substances, adding negatively charged substances), hydrophobic interaction, one-step or multi-step curing, mineralization, encapsulation, etc.

[0051] Furthermore, the water-soluble antigens and / or water-insoluble antigens loaded on the surface of the nanoparticles or microparticles are one layer or multiple layers after loading. When multiple layers of water-soluble antigens and / or water-insoluble antigens are surface-loaded, the layers are separated by modifiers.

[0052] Furthermore, the particle size of the particles used to activate antigen-presenting cells is in the nanometer or micrometer range, which can ensure that the particles are phagocytosed by antigen-presenting cells. In order to improve the phagocytosis efficiency, the particle size should be within an appropriate range. The particle size of the nanoparticles is 1 nm - 1000 nm, more preferably, the particle size is 30 nm - 1000 nm, most preferably, the particle size is 50 nm - 600 nm; the particle size of the microparticles is 1 μm - 1000 μm, more preferably, the particle size is 1 μm - 100 μm, more preferably, the particle size is 1 μm - 10 μm, most preferably, the particle size is 1 μm - 5 μm.

[0053] Dendritic cell vaccine is a type of vaccine. Since DC belongs to live cell products, there are many defects in the storage, transportation, and administration processes of activated DC used as a vaccine. In the present invention, nanoparticles and / or microparticles loaded with whole cell antigens of cells or tissues containing autoimmune disease antigens are first used to activate antigen-presenting cells, so that the antigen-presenting cells are loaded with a broad-spectrum of whole cell antigens. Then, the antigen-presenting cells are processed by mechanical disruption, gradient centrifugation, and / or membrane filtration and / or co-action with particles to prepare a nano-vaccine or a micro-vaccine. The obtained nano-vaccine or micro-vaccine is loaded with antigen epitopes of autoimmune diseases and antigen-presenting cell components. Therefore, it is easy to store, transport, and use. Moreover, after being injected into the human body, it has a certain homing effect, is easily phagocytosed by antigen-presenting cells, and can activate a broad-spectrum and diverse specific regulatory T cells, thereby more and better inhibiting effector T cells in the body.

[0054] By the above scheme, the present invention has at least the following advantages:

[0055] (1) In the vaccine, components of one or more antigen-presenting cells can be integrated with antigen epitopes of autoimmune diseases into a nano-vaccine or a micro-vaccine. A nano-vaccine or a micro-vaccine can contain components of at least one antigen-presenting cell including DC cells, B cells, or macrophages. Therefore, a nano-vaccine or a micro-vaccine can simultaneously have some functions and advantages of one or more antigen-presenting cells.

[0056] (2) The nano-vaccine or the micro-vaccine can contain components of one or more antigen-presenting cells including DC cells. After being injected into the body, it has the characteristic of homing to lymph nodes and can better activate antigen-specific inhibitory immune responses.

[0057] (3) Nanoparticles and / or microparticles loaded with whole cell antigens of cells and / or tissues containing autoimmune disease antigens are used to activate antigen-presenting cells, and then antigen-presenting cells are used to prepare a nano-vaccine. Therefore, the prepared nano-vaccine can be loaded with all antigen epitopes loaded by the nanoparticles and / or microparticles used to activate antigen-presenting cells, because the nano-vaccine can activate a broad-spectrum and diverse regulatory antigen-specific T cells (Treg).

[0058] (4) The nano-vaccine or the micro-vaccine is derived from antigen-presenting cells, and the components are all biocompatible and biodegradable, with good safety.

[0059] (5) After processing antigen-presenting cells with cell activity into a nano-vaccine or a micro-vaccine without any cell activity, the nano-vaccine or the micro-vaccine does not have such harsh requirements for storage, transportation, and injection use as live antigen-presenting cells.

[0060] (6) Based on wrapping the membrane components of antigen-presenting cells on delivery particles, the present invention also loads the membrane components of cells containing self-antigens on the surface of nanoparticles or microparticles, thus endowing the nano-vaccine or micro-vaccine with the ability to load broad-spectrum antigens and the ability to have a cell membrane structure on the surface. Through the effect of surface-loaded biomimetic membranes, some effects that cannot be achieved by particles without biomimetic membranes can be realized.

[0061] The above description is only an overview of the technical solution of the present invention. In order to be able to more clearly understand the technical means of the present invention and to be implemented in accordance with the content of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0063] Figure 1 It is a schematic diagram of the preparation process and application of the nano-vaccine or micro-vaccine of the present invention; wherein, a is a schematic diagram of collecting the water-soluble components and non-water-soluble components in the whole cell antigen after lysing cells or tissues containing self-immune disease antigens, and then preparing nanoparticles or microparticles; b is a schematic diagram of lysing and dissolving the whole cell antigen of cells or tissues containing self-immune disease antigens with a lysate containing a solvent and preparing nanoparticles or microparticles; c is a schematic diagram of using the above particles prepared in a or b to activate antigen-presenting cells, then preparing the antigen-presenting cells into nano-vaccines or micro-vaccines, and using such nano-vaccines to prevent or treat self-immune diseases such as type I diabetes.

[0064] Figures 2 - 15 They are respectively the experimental results when using nano-vaccines or micro-vaccines to prevent or treat self-immune diseases such as type I diabetes in Examples 1-14. Figure 3 、 4 In Examples 1-14, a is the result when preventing or treating self-immune diseases such as type I diabetes, and b and c are the results of using flow cytometry to analyze the proportion of CD8 + and CD4 + regulatory specific T cells in the total CD8 + and CD4 + T cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0065] The following further describes the present invention in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0066] The nano-vaccine or micro-vaccine for preventing or treating autoimmune diseases such as type I diabetes according to the present invention comprises a nano-vaccine or micro-vaccine prepared from antigen-presenting cells activated by nanoparticles and / or microparticles loaded with cells of autoimmune disease antigens and / or whole cell antigens of tumor tissues. The nanoparticles and / or microparticles are loaded with cells of autoimmune disease antigens and / or whole cell antigens of tissues or a mixture thereof. For the preparation of the nano-vaccine or micro-vaccine for preventing or treating autoimmune diseases such as type I diabetes, the preparation process and application fields are as Figure 1 shown.

[0067] When preparing nanoparticles or microparticles loaded with cells containing autoimmune disease antigens and / or whole cell antigens of tumor tissues for activating antigen-presenting cells, the cells or tissues can be lysed and then the water-soluble antigens and water-insoluble antigens can be collected separately and nanoparticle or microparticle systems can be prepared separately; or alternatively, a lysing solution containing a solubilizing agent can be directly used to lyse the cells or tissues and dissolve the whole cell antigens of the cells and nanoparticle or microparticle systems can be prepared. The whole cell antigens of the cells in the present invention can be prepared into nanoparticles or microparticles after being treated by, including but not limited to, inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc. before and / or after lysis; or they can be directly prepared into nanoparticles or microparticles without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment before and / or after cell lysis. In some embodiments of the present invention, the tissue cells are inactivated or (and) denatured before lysis, and in actual use, inactivation or (and) denaturation treatment can also be performed after cell lysis, or inactivation or (and) denaturation treatment can be performed both before and after cell lysis; in some embodiments of the present invention, the inactivation or (and) denaturation treatment method before and / 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 can understand that in actual application, technicians can make appropriate adjustments according to specific situations.

[0068] When using nanoparticles or microparticles to activate antigen-presenting cells in vitro, cytokines and / or antibodies can be used to assist in improving the activation efficiency. The antigen-presenting cells can be derived from autologous or allogeneic sources, or can come from cell lines or stem cells. The antigen-presenting cells can be DC cells, B cells, macrophages or any mixture of the above three, or can be other cells with antigen-presenting functions.

[0069] After the antigen-presenting cells are activated, the antigen-presenting cells are prepared into nano-vaccines or micro-vaccines by mechanical cell disruption followed by methods such as centrifugation and / or membrane filtration and / or co-action with nanoparticles or microparticles. In actual preparation, other methods that can prepare living antigen-presenting cells into nano-vaccines or micro-vaccines without cell activity can also be used.

[0070] In some embodiments, the specific preparation method of preparing antigen-presenting cells into nano-vaccines or micro-vaccines after activating the antigen-presenting cells with nanoparticles or microparticles loaded with cells containing autoimmune disease antigens and / or whole cell antigens of tissues is as follows:

[0071] Step 1, add 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 raw materials for preparing particles.

[0072] In some embodiments, the aqueous solution may contain the components in the cell lysate and an immunosuppressant; the components in the cell lysate are respectively water-soluble antigens or originally water-insoluble antigens dissolved in solvents such as urea or guanidine hydrochloride during preparation. The concentration of the water-soluble antigen or the originally water-insoluble antigen contained in the aqueous solution, that is, the first predetermined concentration, requires the protein polypeptide concentration to be greater than 1 ng / mL, and can load sufficient whole cell antigens to activate related cells. The concentration of the immunosuppressant in the initial aqueous phase is greater than 0.01 ng / mL.

[0073] In some embodiments, the aqueous solution contains the components in the tissue lysate and an immunosuppressant; the components in the tissue lysate are respectively water-soluble antigens or originally water-insoluble antigens dissolved in solvents such as urea or guanidine hydrochloride during preparation. The concentration of the water-soluble antigen or the originally water-insoluble antigen contained in the aqueous solution, that is, the first predetermined concentration, requires the protein polypeptide concentration to be greater than 0.01 ng / mL, and can load sufficient whole cell antigens to activate related cells. The concentration of the immunosuppressant in the initial aqueous phase is greater than 0.01 ng / mL.

[0074] In some embodiments, the raw materials for preparing particles are poly(lactic-co-glycolic acid) (PLGA) or polylactic acid (PLA), and the organic solvent is dichloromethane. Additionally, in some embodiments, the range of the second predetermined concentration of the raw materials for preparing particles is 0.5 mg / mL - 5000 mg / mL, preferably 100 mg / mL.

[0075] In the present invention, PLGA, PLA, or modified PLGA or PLA is selected because this material is a biodegradable material and has been approved by the FDA for use as a pharmaceutical excipient. Research has shown that PLGA and PLA have certain immunomodulatory functions and are thus suitable as excipients for the preparation of nanoparticles or microparticles. In practical applications, appropriate materials can be selected according to actual circumstances.

[0076] In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase. In the present invention, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1:10. During the specific implementation process, the first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the prepared nanoparticles or microparticles.

[0077] Preferably, when the aqueous phase solution is a lysate component solution, the concentration of proteins and polypeptides is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL; when the aqueous phase solution is a lysate component / immunoinhibitor solution, the concentration of proteins and polypeptides is greater than 1 ng / mL, preferably 1 mg / mL to 100 mg / mL, and the concentration of the immunoadjuvant is greater than 0.01 ng / mL, preferably 0.01 mg / mL to 20 mg / mL. In the organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane; the concentration of the organic phase is 0.5 mg / mL to 5000 mg / mL, preferably 100 mg / mL.

[0078] Step 2: Subject the mixture obtained in Step 1 to ultrasonic treatment for more than 2 seconds, or stirring for more than 1 minute, or homogenization treatment, or microfluidic treatment. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm to 1500 rpm and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is greater than 5 W and the time is greater than 0.1 second, for example, 2 to 200 seconds; during homogenization treatment, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used. When using a high-pressure / ultra-high-pressure homogenizer, the pressure is greater than 5 psi, for example, 20 psi to 100 psi. When using a high-shear homogenizer, the rotation speed is greater than 100 rpm, for example, 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, for example, 0.1 mL / min to 100 mL / min. Ultrasonic treatment, stirring, homogenization treatment, or microfluidic treatment is used for nanosizing and / or micronizing. The length of the ultrasonic time, the stirring speed, the pressure and time of the homogenization treatment can control the size of the prepared micro-nano particles. Too large or too small will cause changes in the particle size.

[0079] Step 3: Add the mixture obtained after the treatment in Step 2 into an aqueous solution containing an emulsifier with a third predetermined concentration and a third predetermined volume, and perform ultrasonic treatment for more than 2 seconds, stirring for more than 1 minute, homogenization treatment, or microfluidic treatment. This step adds the mixture obtained in Step 2 into the aqueous solution of the emulsifier and continues ultrasonic treatment or stirring for nanosizing or microsizing. In the present invention, the ultrasonic time is greater than 0.1 second, such as 2 to 200 seconds, the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm, and the stirring time is greater than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm and the stirring time is greater than 1 minute, such as the stirring speed is 50 rpm to 1500 rpm and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50 W to 500 W and the time is greater than 0.1 second, such as 2 to 200 seconds; during homogenization treatment, a high-pressure / ultra-high-pressure homogenizer or a high-shear homogenizer is used. When using a high-pressure / ultra-high-pressure homogenizer, the pressure is greater than 20 psi, such as 20 psi to 100 psi, and when using a high-shear homogenizer, the rotation speed is greater than 1000 rpm, such as 1000 rpm to 5000 rpm; when using microfluidic treatment, the flow rate is greater than 0.01 mL / min, such as 0.1 mL / min to 100 mL / min. Ultrasonic treatment, stirring, homogenization treatment, or microfluidic treatment is carried out for nanosizing or microsizing. The length of the ultrasonic time, the stirring speed, the pressure and time of homogenization treatment can control the size of the prepared nano- or micro-particles. Too large or too small will cause changes in the particle size.

[0080] In some embodiments, the aqueous solution of the emulsifier is an aqueous solution of poly(ethylene-alt-maleic anhydride) (PEMA) or polyvinyl alcohol (PVA), the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg / mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the range of the second predetermined volume to the third predetermined volume is set to be 1:1.1 - 1:1000, and preferably it can be 2:5. During the specific implementation process, in order to control the size of the nano-particles or micro-particles, the ratio of the second predetermined volume to the third predetermined volume can be adjusted. Similarly, the values of the ultrasonic time or stirring time, the volume and concentration of the aqueous solution of the emulsifier in this step are all for obtaining nano-particles or micro-particles with appropriate sizes.

[0081] Step 4: Add the liquid obtained after the treatment in Step 3 into an aqueous solution of an emulsifier with a fourth predetermined concentration and a fourth predetermined volume, and stir until the predetermined stirring conditions are met.

[0082] In this step, the aqueous solution of the emulsifier is a PEMA or PVA solution or other solutions.

[0083] The fourth predetermined concentration is 5 mg / mL. The selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles with appropriate sizes. The selection of the fourth predetermined volume is determined by 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 fourth predetermined volume ranges from 1:1.5 to 1:2000, preferably 1:10. During the specific implementation process, the ratio of the third predetermined volume to the fourth predetermined volume can be adjusted to control the sizes of the nanoparticles or microparticles.

[0084] In the present invention, the predetermined stirring condition for this step is until the organic solvent has completely volatilized, that is, until the dichloromethane in Step 1 has volatilized completely.

[0085] Step 5: After centrifuging the mixture solution processed in Step 4 under the predetermined stirring condition at a rotational speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in an aqueous solution containing a freeze-drying protectant with a fifth predetermined volume and a fifth predetermined concentration, or in a sixth predetermined volume of PBS (or physiological saline).

[0086] In some embodiments of the present invention, when the precipitate obtained in Step 5 is resuspended in a sixth predetermined volume of PBS (or physiological saline), no freeze-drying is required, and subsequent experiments related to the adsorption of cancer cell lysates on the surface of the nanoparticles or microparticles can be directly carried out.

[0087] In some embodiments of the present invention, when the precipitate obtained in Step 5 is resuspended in an aqueous solution containing a freeze-drying protectant, freeze-drying is required, and subsequent experiments related to the adsorption of cancer cell lysates on the surface of the nanoparticles or microparticles are carried out after freeze-drying.

[0088] In the present invention, the freeze-drying protectant is selected as trehalose.

[0089] In the present invention, the fifth predetermined concentration of the freeze-drying protectant in this step is 4% by mass. Such a setting is to ensure that the freeze-drying effect is not affected during subsequent freeze-drying.

[0090] Step 6: After subjecting the suspension containing the freeze-drying protectant obtained in Step 5 to freeze-drying treatment, reserve the freeze-dried substance.

[0091] Step 7: Directly use the suspension containing nanoparticles resuspended in PBS (or physiological saline) with a sixth predetermined volume obtained in Step 5, or directly use the freeze-dried substance containing nanoparticles or microparticles and a freeze-drying protectant obtained by resuspending the product obtained in Step 6 with a sixth predetermined volume of PBS (or physiological saline); or use the above samples after mixing them with a seventh predetermined volume of a water-soluble antigen or a dissolved originally water-insoluble antigen.

[0092] In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is from 1:10,000 to 10,000:1, preferably from 1:100 to 100:1, and most preferably from 1:30 to 30:1.

[0093] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble antigen or the dissolved original non-water-soluble antigen in the cell lysate or tissue lysate is 1 mL. In actual use, the volumes and ratios of the two can be adjusted as needed.

[0094] Step 8: Co-incubate the antigen-presenting cells with the nanoparticles and / or microparticles prepared above for a certain period of time. The tissues and / or cells used to prepare the nanoparticles and / or microparticles and the antigen-presenting cells can be autologous or allogeneic.

[0095] Step 9: Collect the antigen-presenting cells after co-incubation and / or the cultured cells containing self-antigens, such as β cells, which can be washed or unwashed and then treated by ultrasonic treatment, mechanical stirring, homogenization, etc.

[0096] Step 10: Subject the mechanically treated antigen-presenting cells and / or cell samples containing self-antigens to gradient centrifugation, and / or membrane filtration, and / or co-action with nanoparticles or microparticles, etc., to prepare a nano-vaccine or a micro-vaccine.

[0097] In some other embodiments, the method for preparing a nano-vaccine or a micro-vaccine is as follows:

[0098] Steps 1 to 4 are the same as above.

[0099] Step 5: After centrifuging the mixture obtained in Step 4 under the predetermined stirring conditions at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in a solution of the fifth predetermined volume and the fifth predetermined concentration containing water-soluble and / or non-water-soluble antigens of the whole cell antigen of the cells, or resuspend the remaining precipitate in a solution of the fifth predetermined volume and the fifth predetermined concentration containing a mixture of water-soluble and / or non-water-soluble antigens of the whole cell antigen of the cells and an immunosuppressant.

[0100] Step 6: After centrifuging the mixture obtained in Step 5 under the predetermined stirring conditions at a speed greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in a sixth predetermined volume of a solidification treatment reagent or a mineralization treatment reagent, allow it to act for a certain period of time and then centrifuge and wash, and then add a seventh predetermined volume of a substance carrying a positive charge or a negative charge and allow it to act for a certain period of time.

[0101] In some embodiments of the present invention, after the precipitate obtained in step 6 is resuspended in a seventh predetermined volume of charged substance, lyophilization may not be required, and subsequent experiments related to loading cell / tissue lysates on the surface of nanoparticles or microparticles can be directly carried out.

[0102] In some embodiments of the present invention, the precipitate obtained in step 6 is resuspended in an aqueous solution containing a cryoprotectant and then subjected to vacuum drying at room temperature or freeze-vacuum drying. After drying, subsequent experiments related to adsorbing cell lysates on the surface of nanoparticles or microparticles are carried out.

[0103] In the present invention, the cryoprotectant is selected from trehalose, or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the cryoprotectant in this step is 4% by mass. Such a setting is to ensure that the drying effect is not affected during subsequent drying.

[0104] Step 7: After the suspension containing the cryoprotectant obtained in step 6 is dried, the dried substance is reserved for use.

[0105] Step 8: An eighth predetermined volume of the nanoparticle-containing suspension resuspended in PBS (or physiological saline) obtained in step 6, or the dried substance containing nanoparticles or microparticles and the cryoprotectant obtained in step 7 is resuspended directly using an eighth predetermined volume of PBS (or physiological saline) and used directly; or used after being mixed with a ninth predetermined volume of a water-soluble antigen or a water-insoluble antigen.

[0106] In the present invention, the modification and antigen loading steps of steps 5 - 8 can be repeated multiple times to increase the antigen loading amount. Moreover, when adding a positively charged or negatively charged substance, the same charged substance can be added multiple times, or substances with different charges can be added alternately.

[0107] In some embodiments, when the volume of the resuspended nanoparticle suspension is 10 mL, the volume of the water-soluble antigen or the original water-insoluble antigen contained in the cell lysate or tissue lysate is 0.1 - 100 mL. In actual use, the volumes and ratios of the two can be adjusted according to needs.

[0108] Step 9: Co-incubate the antigen-presenting cells with the nanoparticles and / or microparticles prepared above for a certain period of time. The tissue and / or cells used to prepare the nanoparticles and / or microparticles and the antigen-presenting cells can be from autologous or allogeneic sources.

[0109] Step 10: Collect the antigen-presenting cells after co-incubation and / or the cultured cells containing self-antigens, such as β cells, which can be washed or unwashed and then subjected to treatments such as sonication, mechanical stirring, homogenization, etc.

[0110] Step 11: Subject the mechanically treated antigen-presenting cells and / or cell samples containing self-antigens to gradient centrifugation, and / or membrane filtration, and / or co-action with nanoparticles or microparticles, etc., to prepare a nano-vaccine or a micro-vaccine.

[0111] Example 1: Nano-vaccine Based on DC Cell Membrane for the Prevention of Type I Diabetes

[0112] The NIT-1 cell is a mouse β-cell model and can be used as pancreatic islet β-cells. First, lyse the NIT-1 cells to prepare the water-soluble fraction and the water-insoluble fraction of the NIT-1 cells. Then, using the organic polymer material PLA as the nano-particle skeleton material and rapamycin as an immunosuppressant, prepare nanoparticles loaded with the water-soluble fraction and the water-insoluble fraction of β-cells by the solvent evaporation method. Then, use these particles to activate dendritic cells (DCs) and activate the dendritic cells in a way that activates regulatory T cells. Then, after ultrasonic treatment of the dendritic cells, prepare a nano-vaccine to prevent type I diabetes.

[0113] (1) Lysis of β-cells and Collection of Each Fraction

[0114] After culturing NIT-1 β-cells in high-glucose medium, collect the cultured NIT-1 β-cells, remove the medium by centrifugation, resuspend the NIT-1 β-cells with ultrapure water, then freeze them at -20°C to -273°C, add a certain amount of ultrapure water and perform repeated freeze-thaw cycles more than 3 times, accompanied by ultrasonic treatment to disrupt and lyse the cells. After cell lysis, centrifuge the lysate at 3000g for 5 minutes, and take the supernatant as the water-soluble fraction of NIT-1 β-cells that can be dissolved in pure water; add 8M urea (containing 500 mM sodium chloride) to the obtained precipitate to dissolve the precipitate, and the water-insoluble fraction of NIT-1 β-cells that is insoluble in pure water can be converted into a fraction soluble in 8M urea aqueous solution. The above-obtained water-soluble fraction from the cell lysate and the original water-insoluble fraction dissolved in 8M urea are the antigens for preparing the nanoparticles to activate DCs.

[0115] (2) Preparation of Nanoparticles

[0116] In this example, the double emulsion method in the solvent evaporation method was used to prepare nanoparticles. The molecular weight of the polylactic acid (PLA) used as the nanoparticle preparation material was 30 KDa. The immunosuppressant used was rapamycin, and rapamycin was loaded inside the nanoparticles. During the preparation, rapamycin and PLGA were dissolved in the organic phase together, and the emulsifier used was PEMA. The preparation method was as described above. The water-soluble component and the water-insoluble component were loaded on different nanoparticles respectively, and then they were mixed and used together when in use. During the preparation process, the double emulsion method was first used to load the whole cell lysate component and the immunosuppressant inside the nanoparticles. After loading the cell lysate component and the adjuvant inside, 100 mg of the nanoparticles were centrifuged at 10000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h. Before use, 20 mg of the freeze-dried nanoparticles were resuspended in 0.9 mL of PBS, and then mixed with 0.1 mg of the lysate component (60 mg / mL) and allowed to act at room temperature for 3 minutes to obtain the nanoparticles loaded with whole cell antigens both inside and outside. The average particle size of the nanoparticles was 300 nm, and the surface potential was about -3 mV. Approximately 130 μg of protein or polypeptide component was loaded per 1 mg of PLA nanoparticles, and about 0.05 mg of rapamycin was loaded per 1 mg of PLA nanoparticles. The blank nanoparticle preparation material and the preparation method were the same, with a particle size of 300 nm, loading an equal amount of immunosuppressant but not loading any lysate component.

[0117] (3) Preparation of bone marrow-derived dendritic cells (BMDC)

[0118] In this example, the preparation of dendritic cells from mouse bone marrow cells was used as an example to illustrate how to prepare BMDC. First, one 6-8-week-old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The muscle tissues around the bones were carefully removed with scissors and forceps. The two ends of the bones were cut off with scissors, and then PBS solution was drawn with a syringe. The needle was inserted into the bone marrow cavity from both ends of the bone, and the bone marrow was repeatedly rinsed into the culture dish. The bone marrow solution was collected, centrifuged at 400 g for 3 min, and then 1 mL of red blood cell lysate was added to lyse the red blood cells. 3 mL of RPMI 1640 (10% FBS) medium was added to terminate the lysis, centrifuged at 400 g for 3 min, and the supernatant was discarded. The cells were placed in a 10 mm culture dish for culture, using RPMI 1640 (10% FBS) medium, and at the same time recombinant mouse GM-CSF (20 ng / mL) was added, and cultured at 37 °C in 5% CO2 for 7 days. On the 3rd day, the culture flask was gently shaken, and the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL) was supplemented. On the 6th day, half of the medium was replaced. On the 7th day, a small amount of suspended and semi-adherent cells were collected, and by flow cytometry, when CD86 + CD80 + cells were in CD11c+ When the proportion in cells is between 15% and 20%, the induced cultured BMDC can be used for the next experiment.

[0119] (4) Activation of DC

[0120] Incubate nanoparticles loaded with whole cell antigen from β cells (500 μg, including 250 μg of nanoparticles loaded with water-soluble components and 250 μg of nanoparticles loaded with water-insoluble components) or blank nanoparticles (500 μg) + free lysate with BMDC (10 million) in 15 mL of complete RPMI 1640 medium for 96 hours (37 °C, 5% CO2); the cytokine combination in the incubation system includes granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000 U / mL), IL-2 (200 U / mL), IL-4 (500 U / mL), IL-7 (200 U / mL), IL-10 (1000 U / mL).

[0121] (5) Preparation of DC-derived nano-vaccine

[0122] Collect the incubated DC by centrifugation at 400 g for 5 minutes, then wash the cells twice with PBS, resuspend the cells in PBS and sonicate at 7.5 W for 20 minutes. Then centrifuge the sample at 2000 g for 20 minutes and collect the supernatant, centrifuge the supernatant at 7000 g for 20 minutes, collect the supernatant, filter it through a 0.22 μm filter membrane and extrude it to collect the filtrate, then centrifuge the filtrate at 15000 g for 120 minutes, collect and discard the supernatant to collect the precipitate, and resuspend the precipitate in PBS to obtain the nano-vaccine based on DC cell membrane, with the particle size of the nano-vaccine being 120 nanometers. The nano-vaccine prepared using DC activated by nanoparticles loaded with whole cell antigen is nano-vaccine 1; the nano-vaccine prepared using DC activated by blank nanoparticles + free lysate is nano-vaccine 2.

[0123] Alternatively, collect the DC activated in step (4), centrifuge at 400 g for 5 minutes, then wash the cells twice with PBS, and directly use the resuspended cells in PBS as DC vaccine 3.

[0124] (6) Prevention of type 1 diabetes in mice by nano-vaccine or DC vaccine

[0125] In this study, the control groups were the PBS group and the blank nanoparticles + cell lysate group. Female NOD mice at 3 weeks of age were selected for this experiment. In the experiment, 10 NOD mice were in each group. Starting from the third week, 100 μg of nano-vaccine 1, 100 μg of nano-vaccine 2, or 5 million DC vaccines 3 were subcutaneously injected every 7 days for 6 consecutive weeks. The PBS control group was subcutaneously injected with 100 μL of PBS every 7 days starting from the third week for 6 consecutive weeks. The blood glucose of each group of mice was recorded daily starting from the 8th week. Diabetes was considered to have started when the blood glucose was higher than 11.0 mmol·L-1. The incidence of diabetes in NOD mice at different time periods was recorded.

[0126] (7) Experimental results

[0127] NOD mice are type I diabetic model mice. Approximately 70%-85% of female NOD mice will develop type I diabetes after 24 weeks without preventive treatment. As Figure 2 shown, 70%-80% of the mice treated with nano-vaccine 2 prepared from antigen-presenting cells activated by PBS or blank nanoparticles + cell lysate had diabetes after 25 weeks; approximately 50% of the mice treated with live cell DC vaccines loaded with whole cell antigens had type I diabetes; only about 40% of the mice treated with nano-vaccine 1 prepared from antigen-presenting cells activated by nanoparticles loaded with β-cell whole cell antigens had type I diabetes after 25 weeks. This shows that the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with β-cell whole cell antigens described in the present invention has a good preventive effect on type I diabetes, and the preventive effect is better than that of the live vaccine of antigen-presenting cells activated by nanoparticles.

[0128] Example 2 Preparation of nano-vaccine from antigen-presenting cells for the prevention of type I diabetes

[0129] (1) Lysis of β-cells and collection of each component

[0130] After culturing NIT-1 β-cells in high-glucose medium, the cultured NIT-1 β-cells were collected. After centrifugation, the medium was removed, and the NIT-1 β-cells were resuspended in ultrapure water. Then, they were frozen at -20°C to -273°C, and after adding a certain amount of ultrapure water, they were repeatedly frozen and thawed more than 3 times, accompanied by ultrasound to disrupt and lyse the cells. After the cell lysis was completed, the lysate was centrifuged at 2000g for 10 minutes, and the supernatant was taken as the water-soluble component of NIT-1 β-cells soluble in pure water; 10% sodium deoxycholate was added to the obtained precipitate part to dissolve the precipitate part, so that the water-insoluble component of NIT-1 β-cells could be converted into a soluble component in the aqueous solution. After the above-mentioned water-soluble component derived from the cell lysate and the original water-insoluble component dissolved in sodium deoxycholate were mixed at a mass ratio of 1:1, it was the antigen component for preparing the particles activating antigen-presenting cells.

[0131] (2) Preparation of Microparticle System

[0132] In this example, the microparticles were prepared by the double emulsion method in the solvent evaporation method. The PLGA used as the microparticle preparation material had a molecular weight of 24KDa - 38KDa. The immunosuppressive agents used were a mixed immunosuppressive agent of rapamycin and tacrolimus, and the mixed immunosuppressive agent was distributed inside the microparticles. During the preparation, rapamycin, tacrolimus, and PLGA were dissolved in the organic phase together. The preparation method was as described above. During the preparation process, first, the double emulsion method was used to load the β-cell whole cell lysate components and immunosuppressive agents inside the microparticles. After loading the cell lysate components and immunosuppressive agents inside, 100 mg of the microparticles were centrifuged at 7000 g for 10 minutes. After discarding the supernatant, the precipitate was resuspended in 10 mL of ultrapure water containing 4% trehalose and then freeze-dried for 48 h for standby. The average particle size of the obtained microparticles was about 1.5 μm, the surface potential of the microparticle vaccine was about -7 mV, each 1 mg of PLGA microparticle vaccine loaded about 90 μg of protein or polypeptide components, and each 1 mg of PLGA microparticle vaccine loaded 0.025 mg of rapamycin and tacrolimus respectively. The blank microparticles had a particle size of about 1.4 μm. The blank microparticle preparation materials and preparation methods were the same, loading equal amounts of rapamycin and tacrolimus but not loading any lysed water-soluble and water-insoluble components. The preparation methods of the microparticles loaded with several type I diabetes antigen polypeptides were the same. The polypeptides with equal masses loaded were InsulinB 9 - 23, Insulin A 14 - 20, IGRP 206 - 214, and GAD 225 - 244. The average particle size of the particles was about 1.5 μm, the surface potential of the microparticle vaccine was about -7 mV, and each 1 mg of PLGA microparticles loaded about 90 μg of polypeptide components and an equal amount of immunosuppressive agents.

[0133] (3) Preparation of Antigen-Presenting Cells

[0134] This example takes the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDC. First, a 6-8-week-old C57 mouse was sacrificed by cervical dislocation. The tibia and femur of the hind legs were surgically removed and placed in PBS. The muscle tissue around the bones was carefully removed with scissors and forceps. The two ends of the bones were cut off with scissors, and then PBS solution was aspirated with a syringe. The needle was inserted into the bone marrow cavity from both ends of the bone, and the bone marrow was repeatedly rinsed into a culture dish. The bone marrow solution was collected, centrifuged at 400g for 3 min, and then 1 mL of erythrocyte lysate was added to lyse red blood cells. 3 mL of RPMI 1640 (10% FBS) medium was added to terminate the lysis, and the cells were centrifuged at 400g for 3 min, and the supernatant was discarded. The cells were placed in a 10 mm culture dish and cultured using RPMI 1640 (10% FBS) medium. At the same time, recombinant mouse GM-CSF (20 ng / mL) was added, and the cells were cultured at 37 °C and 5% CO2 for 7 days. On the 3rd day, the culture flask was gently shaken, and the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20 ng / mL) was supplemented. On the 6th day, half of the medium was replaced. On the 7th day, a small amount of suspended and semi-adherent cells were collected. By flow cytometry, when the proportion of CD86 + CD80 + cells in CD11c + cells is between 15-20%, the induced BMDC can be used for the next experiment.

[0135] After sacrificing the NOD mouse, the spleen of the mouse was removed, and a single-cell suspension of mouse splenocytes was prepared. CD19 + B cells in the viable splenocytes were separated by magnetic bead sorting (dead cells were labeled with a live-dead cell dye to remove dead cells) and reserved for use.

[0136] (4) Activation of antigen-presenting cells

[0137] The micron-sized particles (500 μg) loaded with whole cell antigen, or polypeptide micron-sized particles (500 μg), or blank micron-sized particles (500 μg) + free lysate were co-incubated with BMDC (5 million) and B cells (5 million) in 15 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contained GM-CSF (2000 U / mL), IL-2 (200 U / mL), IL-7 (1000 U / mL), IL-10 (2000 U / mL), and CD40 antibody (20 ng / mL).

[0138] (5) Preparation of nano-vaccine based on antigen-presenting cells

[0139] The antigen-presenting cells after incubation were collected by centrifugation at 400 g for 5 minutes, and then the cells were washed twice with phosphate-buffered saline (PBS) containing protease inhibitors at 4 °C. The cells were resuspended in PBS water and sonicated at low power (20 W) for 3 minutes at 4 °C. Then the sample was centrifuged at 3000 g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000 g for 15 minutes and the supernatant was collected. Then the supernatant was discarded after centrifugation at 18000 g for 60 minutes and the precipitate was collected. The precipitate was resuspended in PBS and the sample was filtered through a 0.22 μm membrane filter to obtain the nano-vaccine, and the particle size of the nano-vaccine was 120 nm.

[0140] (6) Nano-vaccine prophylaxis of type I diabetes in mice

[0141] Same as Example 1.

[0142] (7) Analysis of antigen-specific regulatory T cells (T reg )

[0143] Female C57BL / 6 mice at 6-8 weeks old were selected as model mice. On days 0, 7, 14, 28, and 42, 100 μg of nano-vaccine or PBS was subcutaneously injected into each mouse. The mice were sacrificed on day 45, and the spleens of the mice were removed and single-cell suspensions of splenocytes were prepared. B cells and T cells were sorted from mouse splenocytes using magnetic bead sorting. 100 μg of micron particles loaded with β-cell whole cell antigen, 5 million B cells, and 1 million T cells were co-incubated in 5 mL of RPMI1640 complete medium for 48 hours (37 °C, 5% CO2). Then the incubated cells were collected and labeled with live / dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody, and FOXP3 antibody, and then the T cell subsets CD4 + CD25 + FOXP3 + The proportion of T cells in CD4 + T cells and CD8+Ly49 + The proportion of T in CD8 + T cells. The β-cell whole cell antigen loaded on the micron particles can be degraded into antigenic epitopes after being phagocytosed by antigen-presenting cell B cells and presented on the surface of antigen-presenting cells. Specific T cells that can recognize the β-cell whole cell antigen can be activated and highly express specific surface markers after recognizing the β-cell whole cell antigen epitopes. The T cells highly expressing specific markers analyzed by flow cytometry are regulatory T cells, that is, T cells that can inhibit effector T cells (T eff ) that recognize and kill β cells. reg .

[0144] (8) Experimental results

[0145] As Figure 3 shown in a, 70%-80% of the mice treated with the nano-vaccine prepared from antigen-presenting cells activated by PBS or blank micro-particles + cell lysate developed type I diabetes after 25 weeks; 50% of the mice treated with the nano-vaccine prepared from antigen-presenting cells activated by micro-particles loaded with antigen polypeptides developed diabetes after 25 weeks. Only about 30% of the mice treated with the micro-vaccine prepared from antigen-presenting cells activated by micro-particles loaded with whole β-cell antigens developed type I diabetes after 25 weeks. In summary, the nano-vaccine described in the present invention has a preventive effect on type I diabetes. This indicates that the types of antigen epitopes presented by DC + B cells activated by micro-particles loaded with antigen polypeptides are limited, so the number of T cell clones contained in the β-cell specific T reg cell system activated by the nano-vaccine prepared therefrom is very small, and the specific T reg cells that can be inhibited and recognize the same antigen eff are also of fewer types. However, the β-cell antigens presented by DC + B cells activated by micro-particles loaded with whole β-cell antigens are more broad-spectrum, so the number of T reg cell clones activated by the nano-vaccine prepared therefrom is also more broad-spectrum, and the specific T reg cells that can be inhibited and recognize the same antigen eff are also of a broad spectrum, and the effect of preventing type I diabetes is better.

[0146] As Figure 3 shown in b, the proportions of CD8 + Ly49 + T cells and CD4 + CD25 + FOXP3 + T cells induced by the nano-vaccine prepared from DC + B cells activated by micro-particles loaded with whole cell antigens respectively account for a significantly higher proportion of CD8 + T cells and CD4 + T cells than those induced by the nano-vaccines prepared from DC + B cells activated by micro-particles loaded with polypeptides and blank micro-particles + free lysate. Thus, it can be seen that the nano-vaccine prepared from antigen-presenting cells activated by micro-particles loaded with whole cell antigens described in the present invention can better activate β-cell specific T eff cells with the ability to recognize and inhibit T reg cells.

[0147] Example 3 Prevention of type I diabetes by micro-vaccines derived from antigen-presenting cells

[0148] In this example, micron particles were prepared from a mixture of whole cell components of mouse islet tissue lysate and whole cell components of β-cell lysate, and then antigen-presenting cells were activated with the micron particles, and micron vaccines were prepared using the antigen-presenting cells.

[0149] (1) Lysis of islets and β-cells and collection of each component

[0150] After sacrificing the mice, mouse islet tissue was collected. The islet tissue was passed through a cell strainer to prepare a single cell suspension, and an appropriate amount of pure water was added, followed by repeated freezing and thawing 5 times, accompanied by ultrasound to disrupt and lyse the cells. The lysate was centrifuged at 3000 g for 3 minutes, and the supernatant was taken as the water-soluble component. The precipitated part was dissolved in an 8M urea aqueous solution (containing 500 mM sodium chloride) to obtain the water-insoluble component dissolved by the solubilizer.

[0151] NIT-1 cells were cultured in high-glucose DMEM complete medium (glucose content was 4 times the normal content) for 24 hours, and 10 μg of thapsigargin was added during the culture process. Then, the cultured NIT-1 cells were collected, the medium was removed by centrifugation, and the NIT-1 cells were resuspended in ultrapure water. Then, a certain amount of ultrapure water was added, followed by repeated freezing and thawing 5 times, accompanied by ultrasound to disrupt and lyse the cells. After the cell lysis was completed, the lysate was centrifuged at 2000 g for 10 minutes, and the supernatant was taken as the water-soluble component of NIT-1 cells soluble in pure water. The precipitated part was dissolved in an 8M urea aqueous solution (containing 500 mM sodium chloride) to obtain the water-insoluble component after dissolution by the solubilizer. High glucose conditions and the use of thapsigargin can both increase the amount of various antigens and insulin granules synthesized by NIT-1 cells, thereby increasing the content of various antigens contained in the cells.

[0152] The water-soluble components derived from β-cell lysate and the water-soluble components derived from islet tissue lysate were mixed at a mass ratio of 1:1 to obtain a water-soluble component mixture; the water-insoluble components derived from β-cell lysate and the water-insoluble components derived from islet tissue lysate were mixed at a mass ratio of 1:1 to obtain a water-insoluble component mixture; the water-soluble component mixture and the water-insoluble component mixture were mixed at a mass ratio of 2:1 to obtain a whole cell component mixture, which contained whole cell antigens related to type I diabetes.

[0153] (2) Preparation of the micron particle system

[0154] In this example, the micron particles were prepared by the double emulsion method of the solvent evaporation method. The PLA used as the micron particle preparation material had a molecular weight of 40KDa. The immunosuppressants used were a mixed immunosuppressant of cyclosporine A and tacrolimus. The substance used to increase lysosomal escape was NH4HCO3. Moreover, both the mixed immunosuppressant and the substance that increased lysosomal escape were distributed inside the micron particles. During preparation, cyclosporine A, tacrolimus, and PLA were dissolved in the organic phase together, and NH4HCO3 was dissolved in the first aqueous phase together with the lysate components. The preparation method was as described above. During the preparation process, the whole cell lysate components, NH4HCO3, and immunosuppressants were first loaded inside the micron particles by the double emulsion method. Then, 100 mg of the micron particles were centrifuged at 6000 g for 10 minutes. After discarding the supernatant, the precipitate was resuspended in 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h for standby. Before use, it was resuspended in 9 mL of PBS and then 1 mL of the lysate component (protein concentration 80 mg / mL) was added and allowed to act at room temperature for 10 min to obtain a micron particle system loaded with whole cell lysate components both inside and outside. The average particle size of the obtained micron particles was about 2.55 μm, the surface potential of the micron vaccine was about -4 mV, about 130 μg of protein or polypeptide components were loaded per 1 mg of PLA micron vaccine, 0.025 mg of cyclosporine A and tacrolimus were loaded per 1 mg of PLA micron vaccine, and 0.05 mg of NH4HCO3 was loaded.

[0155] (3) Preparation of antigen-presenting cells

[0156] In this example, DC cells and B cells derived from peripheral blood were used as antigen-presenting cells. After sacrificing the mice, the peripheral blood of the mice was collected, and the peripheral blood mononuclear cells (PBMC) of the mice were separated by gradient centrifugation. Then, CD19 + B cells and CD11c + DC were sorted from mouse PBMC by flow cytometry. The above B cells and DC were mixed at a ratio of 1:1 by number to obtain the mixed antigen-presenting cells used.

[0157] (4) Activation of antigen-presenting cells

[0158] The micron particles loaded with whole cell antigen (500 μg) and the mixed antigen-presenting cells (a total of 10 million cells, including 5 million DC and 5 million B cells) were co-incubated in 15 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contained cytokine combination 1: TGF-β (2000 U / mL), IL-4 (200 U / mL), IL-7 (1000 U / mL), IL-10 (2000 U / mL).

[0159] Alternatively, incubate micron particles (500 μg) loaded with whole cell antigen with mixed antigen-presenting cells (a total of 10 million cells, including 5 million DCs and 5 million B cells) in 15 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contains cytokine combination 2: TNF-a (2000 U / mL), IL-12 (200 U / mL), IL-6 (1000 U / mL), IL-15 (2000 U / mL).

[0160] Alternatively, incubate mixed antigen-presenting cells (a total of 10 million cells, including 5 million DCs and 5 million B cells) in 15 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contains cytokine combination 1: TGF-β (2000 U / mL), IL-4 (200 U / mL), IL-7 (1000 U / mL), IL-10 (2000 U / mL).

[0161] (5) Preparation of micron vaccine based on antigen-presenting cells or β-cell cell membrane

[0162] Collect the activated mixed antigen-presenting cells (10 million, including 5 million DCs and 5 million B cells) from step (4) by centrifugation at 300 g for 4 minutes, then wash the cells twice with 4 °C phosphate buffer solution (PBS) containing protease inhibitors. Resuspend the cells in PBS water and sonicate at low power (5 W) for 30 minutes. Then filter the sample through membranes with pore sizes of 10 μm, 5 μm, 2 μm, 1 μm, and 0.45 μm in sequence, collect the filtrate, centrifuge the filtrate at 16000 g for 20 minutes, discard the precipitate, and collect the supernatant. Mix the supernatant and the filtrate with the micron particles (80 mg) prepared in step (2), sonicate at 20 W for 3 minutes, incubate for 15 minutes, filter and extrude through a 5-μm filter membrane, collect the filtrate, then centrifuge the filtrate at 8000 g for 15 minutes, discard the supernatant, and collect the precipitate. Resuspend the precipitate in PBS to obtain the micron vaccine. Among them, the micron vaccine prepared using the mixed antigen-presenting cells activated by adding cytokine combination 1 during the co-incubation of micron particles and mixed antigen-presenting cells is micron vaccine 1, with a particle size of 2.60 μm; the micron vaccine prepared using the mixed antigen-presenting cells activated by adding cytokine combination 2 during the co-incubation of micron particles and mixed antigen-presenting cells is micron vaccine 2, with a particle size of 2.60 μm; the micron vaccine prepared using the mixed antigen-presenting cells co-incubated only with cytokine combination 1 without adding any micron particles is micron vaccine 3, with a particle size of 2.60 μm.

[0163] Alternatively, collect the cultured NIT-1 cells (10 million), remove the culture medium after centrifugation, then wash the cells twice with 4°C phosphate-buffered saline (PBS) containing protease inhibitors, resuspend the cells in PBS water, and sonicate them at low power (5W) for 30 minutes. Then, filter the sample through membranes with pore sizes of 10μm, 5μm, 2μm, 1μm, and 0.45μm in sequence, collect the filtrate, centrifuge the filtrate at 16000g for 20 minutes, discard the precipitate, collect the supernatant, mix the supernatant with the micron particles (80mg) prepared in step (2), sonicate at 20W for 3 minutes, co-incubate for 15 minutes, filter and extrude through a 5μm filter membrane, collect the filtrate, then centrifuge the filtrate at 8000g for 15 minutes, discard the supernatant, collect the precipitate, resuspend the precipitate in PBS to obtain micron vaccine 4 with a particle size of 2.60μm.

[0164] Alternatively, simultaneously collect the activated mixed antigen-presenting cells (5 million, including 2.5 million DC cells + 2.5 million B cells) and NIT-1 cells (5 million) from step (4) and mix them, then wash the cells twice with 4°C phosphate-buffered saline (PBS) containing protease inhibitors, resuspend the cells in PBS water, and sonicate them at low power (5W) for 30 minutes. Then, filter the sample through membranes with pore sizes of 10μm, 5μm, 2μm, and 1μm in sequence, collect the filtrate, centrifuge the filtrate at 16000g for 20 minutes, discard the precipitate, collect the supernatant, mix the supernatant with the micron particles (80mg) prepared in step (2), sonicate at 20W for 3 minutes, co-incubate for 15 minutes, filter and extrude through a 5μm filter membrane, collect the filtrate, then centrifuge the filtrate at 8000g for 15 minutes, discard the supernatant, collect the precipitate, resuspend the precipitate in PBS to obtain micron vaccine 5 with a particle size of 2.60μm.

[0165] (6) Prevention of type 1 diabetes in mice by micron vaccines

[0166] The control group in this study was the PBS group. Female NOD mice at 3 weeks of age were selected for this experiment. In the experiment, 10 NOD mice were in each group. Starting from the third week, 100μg of micron vaccine 1, or 100μg of micron vaccine 2, or 100μg of micron vaccine 3, or 100μg of micron vaccine 4, or 100μg of micron vaccine 5, or 100μL of PBS were subcutaneously injected every 7 days for 6 consecutive weeks. The blood glucose of each group of mice was recorded daily starting from the 8th week. When the blood glucose was higher than 11.0mmol·L -1 it was considered that diabetes began to develop. Record the incidence of diabetes in NOD mice at different time periods.

[0167] (7) Analysis of antigen-specific regulatory T cells (T reg )

[0168] Female C57BL / 6 mice at 6 - 8 weeks old were selected as model mice. On day 0, day 7, day 14, day 28, and day 42, each mouse was subcutaneously injected with 100 μg of micron vaccine 1, or micron vaccine 2, or micron vaccine 3, or micron vaccine 4, or micron vaccine 5. On day 45, the mice were sacrificed, and their spleens were removed to prepare single - cell suspensions of splenocytes. B cells and T cells were sorted from the mouse splenocytes using magnetic bead sorting. 100 μg of micron particles loaded with β - cell whole - cell antigen, 5 million B cells, and 1 million T cells were co - incubated in 5 mL of complete RPMI1640 medium for 48 hours (37 °C, 5% CO2). Then the incubated cells were collected and labeled with live - dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody, and FOXP3 antibody, and then the proportion of CD4 + CD25 + FOXP3 + T cells in CD4 + T cells and the proportion of CD8+Ly49 + T in CD8 + T cells were analyzed. The β - cell whole - cell antigen loaded on the micron particles can be degraded into antigenic epitopes after being phagocytosed by antigen - presenting cell B cells and presented on the surface of antigen - presenting cells. Specific T cells that can recognize the β - cell whole - cell antigen can be activated and highly express specific surface markers after recognizing the β - cell whole - cell antigen epitopes. T cells with high expression of specific markers analyzed by flow cytometry are regulatory T cells, that is, they can inhibit effector T cells (T eff ) that recognize and kill β - cells reg .

[0169] (8) Experimental results

[0170] Such as Figure 4As shown in Fig. a, 80% of the mice in the PBS control group developed type I diabetes after 25 weeks. The proportion of mice with type I diabetes in each group treated with micron vaccines was significantly reduced. This indicates that all types of micron vaccines prepared in this example have good preventive effects against type I diabetes. Among them, 40% of the mice in the micron vaccine 2 treatment group developed diabetes after 25 weeks; about 30% of the mice in the micron vaccine 3 treatment group developed type I diabetes after 25 weeks; 10% of the mice in the micron vaccine 1 treatment group developed type I diabetes after 25 weeks; 20% of the mice in the micron vaccine 4 treatment group developed type I diabetes after 25 weeks; and none of the mice in the micron vaccine 5 treatment group developed type I diabetes after 25 weeks. This shows that the particle surface is loaded with the cell membrane components of mixed antigen-presenting cells activated by the particle-loaded whole cell antigen, which can significantly improve the efficacy of the vaccine in preventing type I diabetes; moreover, the particle surface loaded with the cell membrane components of cells containing type I diabetes-related antigens can also improve the efficacy of the micron vaccine in preventing type I diabetes; and when the particle surface is loaded with both the cell membrane components of mixed antigen-presenting cells and the cell membrane components containing antigens, the preventive effect against type I diabetes is the best. Micron vaccine 1 is significantly better than micron vaccine 3, indicating that activating antigen-presenting cells with particles loaded with whole cell antigen before loading the cell membrane components of antigen-presenting cells on the particle surface can significantly improve the efficacy of the finally prepared particles. Moreover, micron vaccine 1 is better than micron vaccine 2, indicating that adding specific cytokines when using particles to activate mixed antigen-presenting cells helps to improve the efficacy of the vaccine prepared from the cell membrane of antigen-presenting cells. In summary, the micron vaccine described in the present invention has a preventive effect against type I diabetes.

[0171] As Figure 4 shown in Figs. b and 4c, the autoantigen-specific T reg induced by micron vaccine 1 is more reg than that of micron vaccine 2 and micron vaccine 3; while the autoantigen-specific T reg induced by micron vaccine 5 is more eff than that of micron vaccine 1. This shows that internal loading of whole cell antigen of cells and surface loading of the cell membrane of activated antigen-presenting cells, especially the cell membrane of mixed antigen-presenting cells, are all beneficial to inducing more autoantigen-specific T

[0172] In this example, micron vaccines are used, and nano vaccines are also applicable. In this example, type I diabetes in autoimmune diseases is taken as an example. In practice, other autoimmune diseases are also applicable.

[0173] This example uses high glucose conditions and thapsigargin to increase the synthesis of various antigens by cells containing autoimmunity disease antigens. In practical applications, any other method to increase the intracellular antigen synthesis amount can also be used, such as methods to increase the intracellular calcium ion content, etc.

[0174] Example 4: Prevention of type I diabetes using a nano-vaccine

[0175] This example uses a mouse type I diabetes model to illustrate how to use an antigen-presenting cell-derived nano-vaccine to prevent autoimmune diseases. In this example, first, pancreatic tissues were lysed to prepare water-soluble antigens and water-insoluble antigens of the pancreatic tissues; then, a nanoparticle system loaded with the water-soluble antigens and water-insoluble antigens of the pancreatic tissues was prepared. In this example, methods of silicification and adding charged substances were used to increase the antigen loading amount, and only one round of mineralization treatment was performed. In this example, antigen-presenting cells were first activated using nanoparticles, and then nano-vaccines were prepared using the antigen-presenting cells.

[0176] (1) Lysis of pancreatic tissues and collection of each component

[0177] After sacrificing the mice, their pancreatic tissues were removed. The pancreatic tissues were cut into pieces and ground, then incubated with collagenase in RPMI 1640 medium for 30 min, and then a single-cell suspension was prepared through a cell strainer, and an appropriate amount of pure water was added and frozen and thawed repeatedly 5 times, accompanied by ultrasonic treatment to disrupt the lysed cells. After the cells were lysed, the lysate was centrifuged at a speed of 5000 g for 5 minutes and the supernatant was taken as the water-soluble antigen soluble in pure water; 10% sodium dodecyl sulfate (SDS) aqueous solution was added to the obtained precipitate part to dissolve the precipitate part, and the water-insoluble antigen could be converted into being soluble in 10% SDS aqueous solution. The water-soluble antigen and the water-insoluble antigen were mixed at a mass ratio of 2:1, which was the antigen raw material source for preparing the particles.

[0178] (2) Preparation of nanoparticles

[0179] In this example, the nanoparticles and the blank nanoparticles used as a control were prepared by the solvent evaporation method and were appropriately modified and improved. During the preparation of the nanoparticles, two modification methods, namely the low-temperature silanization technique and the addition of charged substances, were used to increase the antigen loading amount. The PLA used as the nanoparticle preparation material had a molecular weight of 40KDa, and the immunosuppressant used was fingolimod. Fingolimod and PLA were dissolved in the organic phase. The preparation method was as described above. During the preparation process, the double emulsion method was first used to load the antigen and immunosuppressant inside the nanoparticles. After loading the lysis components inside, 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, and then the nanoparticles were resuspended with 7 mL of PBS and mixed with 3 mL of PBS solution containing cell lysate (60 mg / mL). Then, it was centrifuged at 10,000 g for 20 minutes, and then resuspended with 10 mL of silicate solution (containing 150 mM NaCl, 80 mM tetramethyl orthosilicate, and 1.0 mM HCl, pH 3.0), and fixed at room temperature for 10 min, and then fixed at -80 °C for 24 h. After centrifugal washing with ultrapure water, it was resuspended with 3 mL of PBS containing protamine (5 mg / mL) and polylysine (10 mg / mL) and allowed to act for 10 min, then centrifuged at 10,000 g for 20 min for washing, resuspended with 10 mL of PBS solution containing lysate (50 mg / mL) and allowed to act for 10 min, then centrifuged at 10,000 g for 20 minutes and resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h; before using the particles, they were resuspended with 7 mL of PBS and then 3 mL of lysis solution component (protein concentration 50 mg / mL) was added and allowed to act at room temperature for 10 min to obtain a nanoparticle system modified by freeze-drying silanization and addition of charged substances and loaded with lysate both inside and outside. The average particle size of the nanoparticles was about 350 nm, and the surface potential of the nanoparticles was about -3 mV; each 1 mg of PLA nanoparticles loaded about 260 μg of protein or polypeptide components, and each 1 mg of PLA nanoparticles loaded 0.03 mg of fingolimod inside.

[0180] The preparation materials and preparation methods of the control polypeptide nanoparticles were the same. They were loaded with four β-cell polypeptide antigens of equal mass, and the others were the same as the nanoparticles loaded with whole cell antigens. Each 1 mg of PLA nanoparticles of the polypeptide nanoparticles loaded 0.03 mg of fingolimod, with an average particle size of about 350 nm and a surface potential of the nanoparticles of about -3 mV. The polypeptide antigens loaded were Insulin B 9-23, Insulin A 14-20, IGRP 206-214, and GAD 225-244.

[0181] The particle size of the blank nanoparticles was about 330 nm. The preparation materials and preparation methods of the blank nanoparticles were the same. They were loaded with an equal amount of adjuvant but did not load any cell lysate components.

[0182] (3) Preparation of DCs

[0183] After sacrificing the mice, the lymph nodes of the mice were removed. The lymph nodes of the mice were minced and filtered through a cell sieve to prepare a single-cell suspension of lymph node cells. Then, DCs expressing CD11c were sorted from the single-cell suspension of lymph node cells using flow cytometry. + of DCs.

[0184] (4) Preparation of bone marrow-derived macrophages (BMDMs)

[0185] The C57 mice were anesthetized and sacrificed by cervical dislocation. The mice were disinfected with 75% ethanol, and then a small incision was made on the back of the mice with scissors. The skin was directly torn by hand to the calf joint of the mice, and the foot joints and skin of the mice were removed. The hind limbs were removed along the greater trochanter of the thigh root of the mice with scissors, and the muscle tissue was removed and placed in a culture dish containing 75% ethanol and soaked for 5 min. Then, the culture dish with fresh 75% ethanol was transferred into the laminar flow hood. The ethanol-soaked leg bones were transferred into cold PBS and soaked to wash away the ethanol on the surface of the tibia and femur. This process could be repeated 3 times. The cleaned femurs and tibias were separated, and the two ends of the femurs and tibias were cut off with scissors respectively. A 1 mL syringe was used to aspirate cold induction medium to blow out the bone marrow from the femurs and tibias, and the blowing and washing were repeated 3 times until no obvious red color was seen in the leg bones. The medium containing bone marrow cells was repeatedly pipetted with a 5 mL pipette gun to disperse the cell clumps, and then the cells were filtered through a 70 μm cell filter and transferred into a 15 mL centrifuge tube. The cells were centrifuged at 1500 rpm / min for 5 min, the supernatant was discarded, and the cells were resuspended in erythrocyte lysate and allowed to stand for 5 min, and then centrifuged at 1500 rpm / min for 5 min. The supernatant was discarded, and the cells were resuspended in cold-prepared bone marrow macrophage induction medium (DMEM high-glucose medium containing 15% L929 medium) and plated. The cells were cultured overnight to remove other fast-adhering miscellaneous cells such as fibroblasts. The non-adherent cells were collected and seeded into dishes or cell culture plates according to the experimental design. Macrophage colony-stimulating factor (M-CSF) was used to stimulate the differentiation of bone marrow cells into mononuclear macrophages at a concentration of 40 ng / mL. The cells were cultured for 8 days, and the morphological changes of macrophages were observed under a light microscope. After 8 days, the cells were digested and collected. The cells were incubated with anti-mouse F4 / 80 antibody and anti-mouse CD11b antibody at 4 °C in the dark for 30 min, and then the proportion of successfully induced macrophages was identified using flow cytometry.

[0186] (5) Activation of antigen-presenting cells

[0187] Incubate nanoparticles loaded with whole cell antigen (500 μg), polypeptide nanoparticles (500 μg), or blank nanoparticles (500 μg) + free lysate with the prepared DCs (5 million) and BMDM cells (5 million) in 15 mL of complete RPMI 1640 medium for 24 hours (37 °C, 5% CO2). The incubation system contains granulocyte-macrophage colony-stimulating factor (GM-CSF, 2000 U / mL), IL-2 (200 U / mL), IL-4 (500 U / mL), IL-10 (2000 U / mL), and PD-L1 antibody (10 ng / mL).

[0188] (6) Preparation of antigen-presenting cell-derived nano-vaccine

[0189] Collect the incubated DCs and BMDM by centrifugation at 400 g for 5 minutes, then wash the cells twice with 4 °C phosphate buffer solution (PBS) containing phosphatase inhibitors and protease inhibitors. Resuspend the cells in PBS water and mechanically disrupt the antigen-presenting cells using a tissue homogenizer at 1500 rpm for 10 minutes at 4 °C. Then filter the sample through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm in sequence. After centrifuging the filtrate at 14000 g for 35 minutes, discard the supernatant and collect the precipitate. Resuspend the precipitate in physiological saline containing 4% mannitol and then freeze-dry to obtain the nano-vaccine. The particle size of the nano-vaccine is 260 nanometers.

[0190] (7) Nano-vaccine prepared from antigen-presenting cells for the prevention of type I diabetes

[0191] Same as Example 1.

[0192] (8) Experimental results

[0193] As Figure 5 shown, 70%-80% of the mice treated with the nano-vaccine prepared from antigen-presenting cells activated by PBS or blank microparticles + cell lysate developed type I diabetes after 25 weeks; 60% of the mice treated with the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with antigenic polypeptides developed diabetes after 25 weeks. Only about 30% of the mice treated with the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with whole cell antigen of pancreatic tissue developed type I diabetes after 25 weeks. In summary, the nano-vaccine described in the present invention has a good preventive effect on type I diabetes.

[0194] Example 5 Nano-vaccine based on antigen-presenting cells for the prevention of type I diabetes

[0195] In this example, β-cells were first lysed with 6M guanidine hydrochloride. Then, using PLA as the nanosphere skeleton material and triptolide as the immunosuppressant, nanoparticles loaded with β-cell whole cell antigen were prepared. After activating antigen-presenting cells with the nanoparticles, the cell membranes of the antigen-presenting cells were used to prepare a nano-vaccine for preventing type I diabetes.

[0196] (1) Lysis of β-cells

[0197] The cultured NIT-1 cell line was collected and centrifuged at 400g for 5 minutes, then the supernatant was discarded and washed twice with PBS. Then, the NIT-1 cells were resuspended and lysed with 6M guanidine hydrochloride. After the whole cell antigen of NIT-1 cells was lysed and dissolved in 6M guanidine hydrochloride, it became the antigen raw material source for preparing the nanoparticle system.

[0198] (2) Preparation of nanoparticles

[0199] In this example, the nanoparticles were prepared by the double emulsion method. The PLA used as the nanoparticle preparation material had a molecular weight of 30KDa, and the immunosuppressant used was triptolide. The preparation method was as described above. During the preparation process, the whole cell antigen of NIT-1 cells and triptolide were first loaded inside the nanoparticles by the double emulsion method. Then, 100 mg of the nanoparticles were centrifuged at 13000g for 25 minutes, and then resuspended with 10 mL of ultrapure water containing 4% trehalose and freeze-dried for 48 h for standby. The average particle size of the nanoparticles was about 250 nm, and the surface potential of the nanoparticles was about -4 mV; about 90 μg of protein or polypeptide components were loaded per 1 mg of PLA nanoparticles, and 0.02 mg of triptolide was loaded. These nanoparticles were used as nano-vaccine 3 in mouse animal experiments.

[0200] (3) Preparation of BMDC

[0201] The preparation method was the same as that in Example 1.

[0202] (4) Activation of antigen-presenting cells

[0203] The micron particles loaded with whole cell antigen (500 μg) were co-incubated with BMDC (10 million) in 20 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2). The incubation system contained TGF-β (1000 U / mL), IL-10 (2000 U / mL), IL-4 (500 U / mL) and PD-L1 antibody (10 ng / mL).

[0204] (5) Preparation of nano-vaccine based on the cell membrane of antigen-presenting cells

[0205] After incubation, the BMDCs (10 million) were collected by centrifugation at 400 g for 5 minutes, then washed twice with 4°C phosphate-buffered saline (PBS) containing protease inhibitors. The cells were resuspended in PBS water and sonicated at 4°C (20 W) for 1 minute, and then homogenized using a high-pressure homogenizer (working pressure 5 MPa) for 1 minute. Then the sample was centrifuged at 1000 g for 15 minutes and the supernatant was collected. After centrifugation at 6000 g for 15 minutes, the precipitate was discarded. After collecting the supernatant and centrifuging at 16000 g for 90 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended and mixed with the nanoparticles loaded with whole cell antigen prepared in step (2) (50 mg), sonicated at 10 W for 30 seconds, co-incubated for 15 minutes, filtered and extruded through a 0.45 μm filter membrane, and the filtrate was collected. Then the filtrate was centrifuged at 12000 g for 20 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in physiological saline to obtain Nano-vaccine 1 with a particle size of 260 nm.

[0206] After incubation, the BMDCs (10 million) were collected by centrifugation at 400 g for 5 minutes, then washed twice with 4°C phosphate-buffered saline (PBS) containing protease inhibitors. The cells were resuspended in PBS water and sonicated at 4°C (20 W) for 1 minute, and then homogenized using a high-pressure homogenizer (working pressure 5 MPa) for 1 minute. Then the sample was centrifuged at 1000 g for 15 minutes and the supernatant was collected. After centrifugation at 6000 g for 15 minutes, the precipitate was discarded. After collecting the supernatant and centrifuging at 16000 g for 90 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended with PBS to obtain Nano-vaccine 2 containing only the activated DC cell membrane components, with a particle size of 130 nm.

[0207] (6) The nano-vaccine is used for preventing type I diabetes

[0208] The control group in this study was the PBS group. Female NOD mice at 3 weeks of age were selected for this experiment. In the experiment, there were 10 NOD mice in each group. Starting from the third week, 100 μg of Nano-vaccine 1, or 100 μg of Nano-vaccine 2, or 100 μg of Nano-vaccine 3, or 100 μL of PBS were subcutaneously injected every 7 days for 6 consecutive weeks. The blood glucose of each group of mice was recorded daily starting from the 8th week. Diabetes was considered to have started when the blood glucose was higher than 11.0 mmol·L-1. The incidence of diabetes in NOD mice at different time periods was recorded.

[0209] (7) Experimental results

[0210] As Figure 6As shown, 80% of the PBS-treated mice developed type I diabetes after 25 weeks; 40% of the mice treated with nano-vaccine 2 developed diabetes after 25 weeks; 40% of the mice treated with nano-vaccine 3 developed diabetes after 25 weeks; and 20% of the mice treated with nano-vaccine 1 developed diabetes after 25 weeks. This indicates that nano-vaccine 1 is superior to nano-vaccine 2 and nano-vaccine 3. Both the internal loading of whole cell antigens of cancer cells and the surface loading of the cell membrane components of activated antigen-presenting cells are beneficial to improving the efficacy of nano-vaccines. Nano-vaccine 2 is a nano-vaccine prepared using the DC cell membrane activated by nanoparticle 3, and is a nanoparticle loaded with cell membrane components in a vesicular form; while nano-vaccine 1 is a solid nanoparticle with whole cell antigens loaded internally and the cell membrane of antigen-presenting cells complexed on the surface, which shows that the solid nano-vaccine with whole cell antigens loaded internally and membrane components loaded on the surface has a better effect than the vesicular nano-vaccine that only has antigens loaded on the surface. Since the surface of the activated antigen-presenting cell membrane has a complex of MHC molecules and antigen polypeptides, both nano-vaccine 1 and nano-vaccine 2 are loaded with the complex of MHC molecules and antigen polypeptides on the surface. The nano-vaccine 1 with a membrane structure can directly induce T cells into regulatory T cells (T reg ) with negative regulatory functions, or can be phagocytosed by antigen-presenting cells and the antigens loaded can be degraded and presented by antigen-presenting cells to indirectly activate regulatory T cells (T reg ). In summary, the nano-vaccine described in the present invention has a good preventive effect on type I diabetes.

[0211] Example 6 Prevention of type I diabetes using nano-vaccine

[0212] In this example, first, islet tissues were lysed using 8M urea, and the components of the islet tissue lysate were dissolved. Then, using PLA as the nano-particle skeleton material and cyclophosphamide as an immunosuppressant, nanoparticles loaded with whole cell antigens were prepared. After activating antigen-presenting cells with the nanoparticles, nano-vaccines were prepared using the antigen-presenting cells for the prevention of the autoimmune disease - type I diabetes.

[0213] (1) Lysis of islet tissues

[0214] After sacrificing the mice, the islet tissues of the mice were dissected and separated according to the experimental procedure. The islet tissues were ground and passed through a cell filter, and then an appropriate amount of 8M urea was used to lyse the cells, and 8M urea was used to dissolve the cell lysate. The above is the source of antigen raw materials for preparing the nanoparticle system.

[0215] (2) Preparation of the nanoparticle system

[0216] In this example, the nanoparticles are prepared by the solvent evaporation method. The molecular weight of the PLA used as the nanoparticle preparation material is 20 KDa. The immunosuppressant used is cyclophosphamide, and the lysate component and cyclophosphamide are encapsulated inside the nanoparticles. The preparation method is as described above. During the preparation process, the double emulsion method is first used to load the lysate component and the immunosuppressant inside the nanoparticles. After loading the antigen lysate component and the immunosuppressant inside, 100 mg of the nanoparticles are centrifuged at 12,000 g for 20 minutes, resuspended with 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h to obtain the lyophilized powder for later use. The average particle size of the nanoparticles is about 280 nm, and the surface potential of the nanoparticles is about -3 mV; about 100 μg of protein or polypeptide component is loaded per 1 mg of PLA nanoparticles, and 0.04 mg of cyclophosphamide is loaded per 1 mg of PLA nanoparticles. The blank nanoparticle preparation material and the preparation method are the same, with a particle size of about 280 nm. The blank nanoparticles are loaded with an equal amount of immunosuppressant but do not load any lysate components. The control nanoparticles are loaded with four antigen polypeptides of equal mass (Insulin B 9-23, Insulin A14-20, IGRP 206-214, and GAD 225-244) to replace the lysate component, and the others are the same as the nanoparticles loaded with whole cell antigen. The amount of cyclophosphamide loaded per 1 mg of PLA nanoparticles in the control nanoparticles is 0.04 mg, the particle size is about 280 nm, the surface potential is about -3 mV, and about 100 μg of polypeptide component is loaded per 1 mg of PLA nanoparticles.

[0217] (3) Preparation of DCs and B cells

[0218] After sacrificing the mice, the lymph nodes of the mice are removed, and a single cell suspension of the mouse lymph nodes is prepared. Then, CD11c + DCs and CD19 + B cells are sorted from the single cell suspension of the lymph node cells using flow cytometry.

[0219] (4) Activation of antigen-presenting cells

[0220] The nanoparticles loaded with whole cell antigen (500 μg) or polypeptide nanoparticles (500 μg) are co-incubated with DCs (5 million) and B cells (5 million) in 20 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2), or the nanoparticles loaded with whole cell antigen (500 μg) are co-incubated with DCs (10 million) in 20 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2); the incubation system contains GM-CSF (1000 U / mL), IL-2 (100 U / mL), IL-10 (2000 U / mL), and TGF-β (2000 U / mL).

[0221] (5) Preparation of Nanovaccines Derived from Antigen-Presenting Cells

[0222] After incubation, collect the DCs (5 million) and B cells (5 million) or only the DCs (10 million) by centrifugation at 400 g for 5 minutes. Then wash the cells twice with 4°C phosphate-buffered saline (PBS) containing protease inhibitors. Resuspend the cells in PBS water and disrupt them by stirring at 2000 rpm for 25 minutes using a homogenizer at 4°C. Then centrifuge the sample at 3000 g for 15 minutes and collect the supernatant. Centrifuge the supernatant at 8000 g for 15 minutes and collect the supernatant. Then mix the obtained supernatant with the corresponding nanoparticles (20 mg) for activating antigen-presenting cells prepared in step (2), stir at 2000 RPM for 2 minutes, filter and extrude through a 0.45 μm filter membrane, and collect the filtrate. Then centrifuge the filtrate at 12000 g for 30 minutes, discard the supernatant, and collect the precipitate. Resuspend the precipitate in PBS to obtain the nanovaccine, and the particle size of the nanovaccine is 300 nm.

[0223] (6) Use of Nanovaccines for the Prevention of Type I Diabetes

[0224] Same as Example 1.

[0225] (7) Experimental Results

[0226] As Figure 7 shown, 70%-80% of the mice treated with nanovaccines prepared from antigen-presenting cells activated by PBS or blank microparticles + cell lysates developed type I diabetes after 25 weeks; the ratio of mice with type I diabetes in the nanovaccine-prevented group was significantly reduced. In summary, the nanovaccine described in the present invention has a good preventive effect on type I diabetes. Moreover, the preventive effect of the nanovaccine prepared from DCs and B cells activated by nanoparticles loaded with whole cell antigens is better than that of the nanovaccine prepared from DCs and B cells activated by nanoparticles loaded with four antigen polypeptides. Moreover, the nanovaccine prepared by mixing DCs and B cells activated by nanoparticles loaded with whole cell antigens has a better effect than the nanovaccine prepared from DCs activated by nanoparticles loaded with whole cancer cell antigens, indicating that nanovaccines prepared from multiple antigen-presenting cells activated by nanoparticles have a better effect. This may be because a part of the components in the activated B cells are included in the nanovaccine prepared from antigen-presenting cells, which can assist in enhancing the activation of specific T cells by the nanovaccine.

[0227] Use of the Nanovaccine Prepared from Activated Antigen-Presenting Cells in Example 7 for the Treatment of Type I Diabetes

[0228] In this example, type I diabetes is used as a model to illustrate how to prepare a nano-vaccine using antigen-presenting cells activated by nanoparticles and use this type of vaccine to prevent and treat autoimmune diseases. First, β cells (NIT-1 cells) are lysed to prepare the whole-cell water-soluble fraction and the water-insoluble fraction, and the water-soluble fraction and the water-insoluble fraction are mixed at a mass ratio of 1:1. Then, using PLA as the nano-particle skeleton material and rapamycin and Gusperimus as immunosuppressants, nanoparticles are prepared, and after activating antigen-presenting cells in vitro with these nanoparticles, a nano-vaccine is prepared to treat type I diabetes.

[0229] (1) Lysis of β cells and collection of each component

[0230] After collecting the NIT-1 cell line cultured in cells, an appropriate amount of pure water is added to swell the cells, and then the cells are repeatedly frozen and thawed 5 times, accompanied by ultrasonic treatment to disrupt and lyse the cells. After the cells are lysed, the lysate is centrifuged at a speed greater than 5000g for 5 minutes, and the supernatant is taken as the water-soluble fraction that can be dissolved in pure water; in the obtained precipitate part, an aqueous solution containing 8M urea and 2% arginine is added to dissolve the precipitate part, and the water-insoluble fraction that is insoluble in pure water can be converted into a soluble form in the aqueous solution. Mixing the whole-cell water-soluble fraction and the water-insoluble fraction at a mass ratio of 1:1 is the raw material source for preparing nanoparticles.

[0231] (2) Preparation of nanoparticles

[0232] In this example, the nanoparticles are prepared by the solvent evaporation method. The PLA used as the nanoparticle preparation material has a molecular weight of 20KDa, and the immunosuppressants used are rapamycin and Gusperimus, and the immunosuppressants are distributed inside the nanoparticles. The preparation method is as described above. During the preparation process, the double emulsion method is first used to load the lysate mixture and immunosuppressants inside the nanoparticles. After loading the lysate and adjuvant inside, 100mg of nanoparticles are centrifuged at 10000g for 20 minutes, and then resuspended in 10mL of ultrapure water containing 4% trehalose and freeze-dried for 48h. Before use, 20mg of nanoparticles are resuspended in 0.9mL of PBS, and incubated at room temperature for 5 minutes after mixing with 0.1mL of a sample containing the lysate mixture (80mg / mL) and then can be used. The average particle size of these nanoparticles is about 280nm, and the surface potential of the nanoparticles is about -3mV; about 140μg of protein or polypeptide components are loaded per 1mg of PLGA nanoparticles, and each 1mg of PLGA nanoparticles contains 0.03mg of rapamycin and Gusperimus respectively.

[0233] (3) Preparation of antigen-presenting cells

[0234] After sacrificing C57BL / 6, collect the mouse peripheral blood, isolate peripheral blood mononuclear cells (PBMC) from the peripheral blood, and then use flow cytometry to sort CD11c from PBMC+ DC. In this example, both DC and BMDM are used as antigen-presenting cells simultaneously. The preparation method of BMDM is the same as that in Example 4.

[0235] (4) Activation of antigen-presenting cells

[0236] The nanoparticles (1000 μg) loaded with whole cell antigen of the cells are co-incubated with DC (5 million) and BMDM (5 million) derived from peripheral blood in 20 mL of RPMI1640 complete medium for 48 hours (37 °C, 5% CO2), or the nanoparticles (500 μg) loaded with whole cell antigen of cancer cells are co-incubated with DC (10 million) derived from peripheral blood in RPMI1640 complete medium for 72 hours (37 °C, 5% CO2); the incubation system contains GM-CSF (1000 U / mL), IL-2 (100 U / mL), IL-10 (2000 U / mL), and IL-4 (500 U / mL).

[0237] (5) Preparation of nanoparticle vaccines derived from antigen-presenting cells

[0238] The incubated DC (5 million) and BMDM (5 million) derived from peripheral blood or only DC (10 million) derived from peripheral blood are collected by centrifugation at 400 g for 5 minutes, then washed twice with 4 °C phosphate buffer solution (PBS) containing protease inhibitors, the cells are resuspended in PBS water and then processed in a high-pressure homogenizer (5000 bar) for 5 minutes. Then the sample is centrifuged at 2000 g for 15 minutes and the supernatant is collected, the supernatant is centrifuged at 8000 g for 15 minutes and the supernatant is collected, the supernatant is co-incubated with the corresponding nanoparticles (60 mg) prepared in step (2) at room temperature for 1 hour, filtered and extruded through a 0.45 μm filter membrane, and the filtrate is collected. Then it is centrifuged at 13000 g for 20 minutes, the supernatant is discarded, and the precipitate is collected. The precipitate is resuspended in PBS to obtain the nanoparticle vaccine, and the particle size of the nanoparticle vaccine is 320 nm.

[0239] (6) Treatment of type 1 diabetes with nanoparticle vaccines

[0240] Female NOD mice at 25 weeks of age with diabetes onset are selected for this experiment. In the experiment, there are 10 NOD mice in each group. Starting from day 0, each mouse is subcutaneously injected with 100 μg of nanoparticle vaccine or PBS every 3 days for 6 consecutive times. The blood glucose of each mouse is recorded every day. Diabetes onset is defined as blood glucose higher than 11.0 mmol·L-1, and diabetes remission and cure are defined as blood glucose lower than 11.0 mmol·L-1 for three consecutive days. The diabetes onset situation of NOD mice at different time periods is recorded.

[0241] (7) Experimental results

[0242] As Figure 8 shown, the blood glucose of mice in the PBS control group was always higher than normal. In the nano-vaccine treatment group, some mice with type I diabetes were cured. Moreover, the nano-vaccine prepared by mixing activated DCs and macrophages had a better effect than the nano-vaccine prepared by DCs activated by nanoparticles, indicating that the nano-vaccine prepared by multiple antigen-presenting cells activated by nanoparticles had a better effect. In summary, the nano-vaccine prepared by the antigen-presenting cells described in the present invention has a good therapeutic effect on type I diabetes.

[0243] Example 8 Nano-vaccine Prepared by Activated Antigen-Presenting Cells for the Treatment of Type I Diabetes

[0244] In this example, type I diabetes was used as a model to illustrate how to use nanoparticles loaded with whole β-cell antigens to activate antigen-presenting cells and then use the nano-vaccine prepared by antigen-presenting cells to treat type I diabetes.

[0245] (1) Lysis of β-cells and Collection of Each Component

[0246] The cultured NIT-1 cells were collected. After lysing the NIT-1 cells in an 8M urea aqueous solution (containing 500 mM sodium chloride), the lysate components were dissolved in an 8M urea aqueous solution, which was the antigen source for preparing nanoparticles.

[0247] (2) Preparation of Nanoparticles

[0248] In this example, the nanoparticles were prepared by the double emulsion method. The PLGA with a molecular weight of 24KDa - 38KDa was used as the nanoparticle preparation material, and the inhibitors used were rapamycin and everolimus. After loading the lysis components and immunosuppressants internally, 100 mg of the nanoparticles were centrifuged at 10,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h for standby. The average particle size of the nanoparticles was about 360 nm, and the surface potential of the nanoparticles was about -5 mV; about 80 μg of protein and polypeptide components were loaded per 1 mg of PLGA nanoparticles, and 0.02 mg of rapamycin and everolimus were loaded per 1 mg of PLGA nanoparticles.

[0249] The preparation materials and methods of the blank nanoparticles were the same as above, with a particle size of about 350 nm, only loaded with an equal amount of adjuvant but no lysis components.

[0250] (3) Preparation of DCs

[0251] DC is a mixed DC of DCs derived from peripheral blood and BMDCs. The preparation method of BMDCs is the same as above. After sacrificing C57BL / 6, mouse peripheral blood is collected, peripheral blood mononuclear cells (PBMCs) are isolated from the peripheral blood, and then CD11c + DCs are sorted from PBMCs using flow cytometry.

[0252] (4) Activation of antigen-presenting cells

[0253] The nanoparticles loaded with whole cell antigen (800 μg) or blank nanoparticles (800 μg) + an equal amount of free lysate are co-incubated with DCs derived from peripheral blood (8 million) and BMDCs (8 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contains IL-2 (100 U / mL), IL-10 (2000 U / mL), IL-4 (1000 U / mL), and TGF-β (1000 U / mL).

[0254] (5) Preparation of antigen-presenting cell-derived nano-vaccines

[0255] After incubation, DCs derived from peripheral blood (8 million) and BMDCs (8 million) are collected by centrifugation at 400 g for 5 minutes, and then the cells are washed twice with 4 °C phosphate buffer solution (PBS) containing protease inhibitors. The cells are resuspended in PBS water and sonicated at 4 °C at low power (20 W) for 1 minute and then processed with a homogenizer at 1000 rpm for 3 minutes. Then the sample is centrifuged at 3000 g for 15 minutes and the supernatant is collected. The supernatant is centrifuged at 8000 g for 15 minutes and the supernatant is collected. The supernatant is sonicated with the corresponding nanoparticles (50 mg) prepared in step 2 of Example 1 and DSPE-PEG-CD32 monoclonal antibody at 10 W for 2 minutes, filtered and extruded through a 0.45 μm filter membrane, and the filtrate is collected. Then the filtrate is centrifuged at 15000 g for 30 minutes, the supernatant is discarded, and the precipitate is collected. The precipitate is resuspended in PBS to obtain nano-vaccine 1. Alternatively, the supernatant is sonicated with the corresponding nanoparticles (50 mg) prepared in step 2 of Example 1 at 10 W for 2 minutes, filtered and extruded through a 0.45 μm filter membrane, and the filtrate is collected. Then the filtrate is centrifuged at 15000 g for 30 minutes, the supernatant is discarded, and the precipitate is collected. The precipitate is resuspended in PBS to obtain nano-vaccine 2. The particle sizes of both nano-vaccine 1 and nano-vaccine 2 are 380 nanometers.

[0256] (6) Treatment of type I diabetes with nano-vaccines

[0257] Same as Example 7.

[0258] (7) Experimental results

[0259] AsFigure 9 As shown, the blood glucose levels of mice treated with the nano-vaccines prepared from antigen-presenting cells activated by PBS control and blank nanoparticles were always higher than normal. However, in the treatment group of the nano-vaccines prepared from antigen-presenting cells activated by nanoparticles loaded with whole-cell antigens, some mice with type I diabetes were cured. Moreover, the nano-vaccine with the CD32 monoclonal antibody targeting head had a better effect than the nano-vaccine without the targeting head. In summary, the nano-vaccine prepared from the antigen-presenting cells described in the present invention has a good therapeutic effect on type I diabetes. In the nano-vaccine of this example, CD32 monoclonal antibody was used as the targeting head for active targeting. In practical applications, any targeting head with the ability to target target cells, such as mannose, mannan, CD205 monoclonal antibody, CD19 monoclonal antibody, etc., can also be used.

[0260] Example 9 Prevention of Type I Diabetes with Antigen-Presenting Cell-Based Nano-Vaccines

[0261] In this example, mouse pancreatic tissue was used to illustrate how to prepare micron particles loaded with whole-cell antigens of pancreatic tissue containing β cells, and then use these micron particles to activate antigen-presenting cells and prepare the antigen-presenting cells into nano-vaccines, and apply this nano-vaccine to prevent type I diabetes. The pancreas contains mouse β cells, which can be used as a source of islet β cells to prepare vaccines. First, the pancreatic tissue of mice was removed, and then the water-soluble fraction and the water-insoluble fraction of the whole cells of the obtained tissue were prepared respectively. Then, using the organic polymer material PLGA as the nano-particle skeleton material, and using mRNA encoding TGF-β and rapamycin as immunosuppressants, micron particles loaded with whole-cell antigens were prepared by the solvent evaporation method, and nano-vaccines were prepared from the antigen-presenting cells activated by these micron particles.

[0262] (1) Collection of Whole-Cell Antigens from Pancreatic Tissue

[0263] After sacrificing BALB / c mice, their pancreatic tissue was removed. The pancreatic tissue was cut into pieces and ground, and an appropriate amount of pure water was added through a cell filter, and the mixture was repeatedly frozen and thawed and ultrasonically treated at least 8 times. After the tissue cells were lysed, the tissue cell lysate was irradiated with ultraviolet light for 15 minutes, then the lysate was heated at 95 °C for 10 minutes, and then the tissue lysate was centrifuged at a speed of more than 3000 RPM for 5 minutes, and the supernatant was taken as the water-soluble fraction that was soluble in pure water in the tissue cells; 8M urea aqueous solution was added to the obtained precipitate to dissolve the precipitate, and the original water-insoluble fraction could be converted into a fraction soluble in 8M urea aqueous solution. The water-soluble fraction and the original water-insoluble fraction dissolved in 8M urea were mixed at a mass ratio of 1:1, which was the antigen source for preparing the micron particles that activate antigen-presenting cells.

[0264] (2) Preparation of Micron Particles for Activating Antigen-Presenting Cells

[0265] In this example, the multiple emulsion method in the solvent evaporation method was used to prepare micron particles. The molecular weight of the PLGA used as the micron particle preparation material was 24KDa - 38KDa. The immunosuppressants used were mRNA encoding TGF-β and rapamycin. The mRNA encoding TGF-β and rapamycin were distributed inside the micron particles. During preparation, the mRNA was dissolved in the aqueous phase while rapamycin was dissolved in the organic phase. The preparation method was as described above. The average particle size of the micron particles was about 1.5 μm, the surface potential Zetapotential was about -8 mV, about 90 μg of protein or polypeptide components were loaded per 1 mg of PLGA micron particles, and 0.01 mg of mRNA was loaded per 1 mg of PLGA micron particles. The particle size of the blank micron particles was about 1.5 μm, and the blank micron particles loaded an equal amount of mRNA but no lysate components.

[0266] (3) Preparation of BMDC and B cells

[0267] The preparation of BMDC was the same as in Example 1. The preparation method of B cells derived from mouse peripheral blood was the same as above.

[0268] (4) Activation of antigen-presenting cells

[0269] The micron particles loaded with whole cell antigen (1000 μg) or blank micron particles (1000 μg) were co-incubated with BMDC (10 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2); or the micron particles loaded with whole cell antigen (1000 μg) were co-incubated with B cells (10 million) in 15 mL of high-glucose DMEM complete medium for 72 hours (37 °C, 5% CO2); the incubation system contained IL-10 (2000 U / mL), IL-2 (500 U / mL), IL-4 (200 U / mL), and IL-13 (200 U / mL).

[0270] (5) Preparation of nano-vaccine based on antigen-presenting cells

[0271] The incubated DC or B cells were collected by centrifugation at 400 g for 5 minutes, then washed twice with 4 °C phosphate buffer solution (PBS) containing protease inhibitors. The cells were resuspended in PBS water and sonicated at 4 °C at low power (10 W) for 10 minutes. Then the sample was filtered through membranes with pore sizes of 10 μm, 5 μm, 2 μm, 1 μm, and 0.45 μm in sequence, and the filtrate was collected. Then, after centrifugation at 14000 g for 40 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in PBS to obtain the nano-vaccine, and the particle size of the nano-vaccine was 160 nm.

[0272] (6) Prevention of type I diabetes with nano-vaccine

[0273] Same as Example 1.

[0274] (7) Experimental results

[0275] As Figure 10 shown, compared with the mice treated with the nano-vaccines prepared from antigen-presenting cells activated by PBS and blank microparticles + cell lysates, the proportion of mice suffering from type I diabetes treated with the nano-vaccines prepared from antigen-presenting cells activated by microparticles loaded with whole cell antigens was significantly reduced. Moreover, the nano-vaccine prepared from DCs activated by microparticles had a better effect than the nano-vaccine prepared from B cells activated by microparticles. Thus, it can be seen that the nano-vaccine described in the present invention has a preventive effect on type I diabetes, and there are differences in the effects of nano-vaccines prepared from different antigen-presenting cells.

[0276] The nano-vaccine prepared from antigen-presenting cells activated in Example 10 is used for preventing type I diabetes

[0277] In this example, type I diabetes is used as a model to illustrate how to use the nano-particles loaded with β-cell whole cell antigens to activate antigen-presenting cells, and then use the nano-vaccines prepared from the antigen-presenting cells to prevent type I diabetes. Moreover, by appropriately treating the β-cells before lysis, the antigen content can be increased, thereby improving the efficiency of antigen-loaded particles in activating antigen-presenting cells.

[0278] (1) Lysis of β-cells and collection of each component

[0279] Collect the cultured NIT-1 cells, remove the culture medium after centrifugation, resuspend the NIT-1 cells with ultrapure water, then freeze them at -20 °C to -273 °C, add a certain amount of ultrapure water and repeatedly freeze-thaw 8 times, accompanied by ultrasonic treatment to disrupt and lyse the cells. After the cells are lysed, centrifuge the lysate at a speed of 2000 g for 15 minutes and take the supernatant, which is the water-soluble component of NIT-1 cells soluble in pure water; add an 8 M urea (containing 500 mM sodium chloride) aqueous solution to the obtained precipitate to dissolve the precipitate, and the water-insoluble component of NIT-1 cells that is insoluble in pure water can be converted into a soluble form in the 8 M urea aqueous solution. Mix the above water-soluble component and water-insoluble component at a mass ratio of 1:1, which is the antigen source for preparing the control nanoparticles.

[0280] Double the glucose content in the high-glucose medium, and simultaneously add 0.05 μM / L Thapsigargin to the medium and incubate NIT-1 cells for 12 hours. Increasing the glucose content or adding Thapsigargin can increase the antigen content in β cells. Then, collect the cultured NIT-1 cells, remove the medium by centrifugation, resuspend the NIT-1 cells with ultrapure water, then add a certain amount of ultrapure water and freeze-thaw 8 times repeatedly, accompanied by ultrasonic treatment to disrupt and lyse the cells. After the cells are lysed, centrifuge the lysate at 2000 g for 15 minutes and take the supernatant, which is the water-soluble fraction of NIT-1 cells soluble in pure water; add an aqueous solution of 8 M urea (containing 500 mM sodium chloride) to the obtained precipitate to dissolve the precipitate, and the water-insoluble fraction of NIT-1 cells can be converted into a fraction soluble in an 8 M urea aqueous solution. Mix the above water-soluble fraction and water-insoluble fraction at a mass ratio of 1:1, which is the antigen source for preparing nanoparticles that activate antigen-presenting cells.

[0281] (2) Preparation of nanoparticles

[0282] In this example, nanoparticles and control nanoparticles were prepared by the double emulsion method. The PLGA used for nanoparticle preparation had a molecular weight of 24 KDa - 38 KDa, and the inhibitors used were rapamycin and everolimus. After internal loading of the lysed components and immunosuppressive agents, 100 mg of nanoparticles were centrifuged at 12000 g for 20 minutes, resuspended with 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 h for later use. The average particle size of these nanoparticles was about 260 nm, and the surface potential of the nanoparticles was about -5 mV; each 1 mg of PLGA nanoparticles loaded approximately 80 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles loaded 0.02 mg of rapamycin and everolimus respectively.

[0283] (3) Preparation of DC

[0284] DC is a mixed DC derived from peripheral blood DC and BMDC. The preparation methods of the two are the same as above.

[0285] (4) Activation of antigen-presenting cells

[0286] Incubate the nanoparticles loaded with whole cell antigen (800 μg) with DC derived from peripheral blood (8 million) and BMDC (8 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contains IL-13 (500 U / mL), IL-10 (2000 U / mL), IL-4 (1000 U / mL), and TGF-β (2000 U / mL).

[0287] (5) Preparation of nanoparticle vaccines derived from antigen-presenting cells

[0288] Peripheral blood-derived DCs (8 million) and BMDCs (8 million) after incubation were collected by centrifugation at 400 g for 5 minutes, and then the cells were washed twice with 4°C phosphate-buffered solution (PBS) containing protease inhibitors. After resuspending the cells in PBS water, they were sonicated at low power (20 W) for 1 minute at 4°C and then processed with a homogenizer at 1000 rpm for 3 minutes. Then the samples were centrifuged at 3000 g for 15 minutes and the supernatant was collected. The supernatant was centrifuged at 8000 g for 15 minutes and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane and the filtrate was collected. The filtrate was stirred with the corresponding nanoparticles (30 mg) prepared in step 2 of Example 1 and DSPE-PEG-CD32 monoclonal antibody (30 μg) at 1200 RPM for 2 minutes, then filtered through a 0.45 μm filter membrane and the filtrate was collected. Then the filtrate was centrifuged at 15000 g for 30 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in PBS to obtain the nano-vaccine, and the particle size of the nano-vaccine was 280 nm.

[0289] (6) Use of the nano-vaccine for the prevention of type I diabetes

[0290] Same as Example 1.

[0291] (7) Experimental results

[0292] As Figure 11 shown, compared with the mice in the PBS control group, the proportion of mice with type I diabetes treated with the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with whole cell antigen was significantly reduced. Moreover, the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles prepared with β-cells treated with high glucose and added chemicals had a better effect than the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles prepared with β-cells without the above treatment. Thus, it can be seen that the nano-vaccine described in the present invention has a preventive effect on type I diabetes, and appropriately treating β-cells to increase the antigen content helps to improve the efficacy of the nano-vaccine prepared from activated antigen-presenting cells.

[0293] Use of the nano-vaccine prepared from antigen-presenting cells activated in Example 11 for the prevention of type I diabetes

[0294] (1) Lysis of β-cells and collection of each component

[0295] Collect the cultured NIT-1 cells. After centrifugation, remove the culture medium, resuspend the NIT-1 cells with ultrapure water, then freeze them at -20°C to -273°C. Add a certain amount of ultrapure water and perform repeated freeze-thaw cycles 8 times, accompanied by ultrasonic treatment to disrupt and lyse the cells. After cell lysis, add nuclease (2 mg / mL) and incubate at 37°C for 30 minutes, then inactivate the nuclease by incubating at 95°C for 5 minutes. Centrifuge the lysate at a speed greater than 100 g for more than 1 minute and take the supernatant, which is the water-soluble fraction of NIT-1 cells soluble in pure water; add an 8 M urea (containing 500 mM sodium chloride) aqueous solution to the obtained precipitate to dissolve the precipitate, thus converting the water-insoluble fraction of NIT-1 cells into a fraction soluble in the 8 M urea aqueous solution. Mix the above water-soluble fraction and water-insoluble fraction at a mass ratio of 1:1, which is the antigen source for preparing the control nanoparticles.

[0296] (2) Preparation of nanoparticles

[0297] In this example, the nanoparticles and the control nanoparticles are prepared by the double emulsion method. The PLGA used for preparing the nanoparticles has a molecular weight of 24 KDa - 38 KDa, and the inhibitors used are rapamycin and tacrolimus. After internally loading the lysed components and the immunosuppressive agents, centrifuge 100 mg of the nanoparticles at 12,000 g for 20 minutes, resuspend them with 10 mL of ultrapure water containing 4% trehalose, and then freeze-dry for 48 h for standby. The average particle size of the nanoparticles is about 250 nm, and the surface potential of the nanoparticles is about -5 mV; each 1 mg of PLGA nanoparticles loads about 80 μg of protein and polypeptide components, and each 1 mg of PLGA nanoparticles loads 0.02 mg of rapamycin and tacrolimus respectively.

[0298] (3) Preparation of DC

[0299] DC is a mixed DC of DC derived from peripheral blood and BMDC. The preparation methods of the two are the same as above.

[0300] (4) Activation of antigen-presenting cells

[0301] The nanoparticles loaded with whole cell antigen (800 μg) were co-incubated with peripheral blood-derived DCs (10 million) and BMDCs (10 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained IL-13 (500 U / mL), IL-10 (2000 U / mL), IL-4 (1000 U / mL), and TGF-β (2000 U / mL). Alternatively, the nanoparticles loaded with whole cell antigen (800 μg) were co-incubated with peripheral blood-derived DCs (8 million) and BMDCs (8 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2), but no cytokines were added to the incubation system.

[0302] (5) Preparation of antigen-presenting cell-derived nano-vaccine

[0303] After incubation, BMDCs or BMDMs were collected by centrifugation at 400 g for 5 minutes, and then washed three times by centrifugation at 1200 rpm for 3 minutes in 30 mM pH 7.0 Tris-HCl buffer containing 0.0759 M sucrose and 0.225 M mannitol. Then, the antigen-presenting cells were mechanically disrupted by sonication in the presence of phosphatase inhibitors and protease inhibitors. The cell membranes obtained after centrifugation were washed with a solution of 10 mM pH 7.5 Tris-HCl and 1 mM EDTA. Then, the sample was filtered successively through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm. After centrifugation of the filtrate at 12000 g for 25 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in physiological saline containing 4% mannitol and freeze-dried to obtain the nano-vaccine, and the particle size of the nano-vaccine was 260 nm.

[0304] (6) Use of the nano-vaccine for the prevention of type I diabetes

[0305] Same as Example 1.

[0306] (7) Experimental results

[0307] As Figure 12As shown, compared with the mice in the PBS control group, the proportion of mice with type I diabetes treated with the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with whole cell antigen was significantly reduced. Moreover, the nano-vaccine prepared from antigen-presenting cells activated by adding cytokines or antibodies during the process of nanoparticle-activated antigen-presenting cells had better effects than the nano-vaccine prepared from antigen-presenting cells without adding any cytokines or antibodies during the process of nanoparticle-activated antigen-presenting cells. Thus, it can be seen that the nano-vaccine described in the present invention has a preventive effect on type I diabetes, and the addition of cytokines and / or antibodies during the activation process of antigen-presenting cells helps to improve the efficacy of the nano-vaccine prepared from the activated antigen-presenting cells.

[0308] Example 12 Nano-vaccine Prepared from Activated Antigen-Presenting Cells for Preventing Type I Diabetes

[0309] (1) Lysis of β-cells and Collection of Each Component

[0310] Collect the cultured NIT-1 cells, remove the culture medium after centrifugation, resuspend the NIT-1 cells with ultrapure water, then freeze them at -20 °C to -273 °C, add a certain amount of ultrapure water and freeze-thaw them 8 times repeatedly, accompanied by ultrasonic treatment to disrupt and lyse the cells. After the cells are lysed, add nuclease (2 mg / mL) and incubate at 37 °C for 30 minutes, then inactivate the nuclease at 95 °C for 5 minutes. Centrifuge the lysate at a speed greater than 100 g for more than 1 minute and take the supernatant, which is the water-soluble component of NIT-1 cells soluble in pure water; add an 8 M urea (containing 500 mM sodium chloride) aqueous solution to the obtained precipitate to dissolve the precipitate, and the water-insoluble component of NIT-1 cells can be converted into a soluble component in the 8 M urea aqueous solution. Mix the above water-soluble component and water-insoluble component at a mass ratio of 1:1 as the antigen source for preparing the control nanoparticles.

[0311] (2) Preparation of Nanoparticles

[0312] In this example, the nanoparticles and control nanoparticles were prepared by the double emulsion method. The PLGA with a molecular weight of 24KDa - 38KDa was used as the material for preparing the nanoparticles, and the inhibitors used were rapamycin and interleukin-2. The KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) was used as the lysosome escape substance. After internal loading of the lysis components, lysosome escape substance and immunosuppressant, 100 mg of the nanoparticles were centrifuged at 12,000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and then freeze-dried for 48 h for standby. The average particle size of the nanoparticles was about 250 nm, and the surface potential of the nanoparticles was about -5 mV; about 80 μg of protein and polypeptide components were loaded per 1 mg of PLGA nanoparticles, 0.02 mg of rapamycin and interleukin-2 were loaded per 1 mg of PLGA nanoparticles, and 0.03 mg of KALA polypeptide was loaded. The preparation material and method of the control nanoparticles were the same, with an average particle size of about 250 nm and a surface potential of about -5 mV; about 80 μg of protein and polypeptide components were loaded per 1 mg of PLGA nanoparticles, 0.03 mg of rapamycin and interleukin-2 were loaded per 1 mg of PLGA nanoparticles, and no substance that increased lysosome escape was loaded.

[0313] (3) Preparation of DC

[0314] The DC was a mixed DC of DC derived from peripheral blood and BMDC. The preparation methods of the two were the same as above.

[0315] (4) Activation of antigen-presenting cells

[0316] The nanoparticles loaded with whole cell antigen (800 μg) were co-incubated with DC derived from peripheral blood (8 million) and BMDC (8 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained IL-13 (500 U / mL), IL-10 (2000 U / mL), IL-4 (1000 U / mL) and TGF-β (2000 U / mL).

[0317] (5) Preparation of nanoparticle vaccines derived from antigen-presenting cells

[0318] After incubation, BMDC or BMDM were collected by centrifugation at 400 g for 5 minutes, and then washed three times by centrifugation at 1200 rpm for 3 minutes in 30 mM Tris-HCl buffer (pH 7.0) containing 0.0759 M sucrose and 0.225 M mannitol. Then, the antigen-presenting cells were mechanically disrupted by sonication in the presence of phosphatase inhibitors and protease inhibitors. The cell membranes obtained after centrifugation were washed with a solution of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. Then, the sample was successively filtered through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, and 0.45 μm. After centrifugation of the filtrate at 12000 g for 25 minutes, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in physiological saline containing 4% mannitol and freeze-dried to obtain the nano-vaccine, and the particle size of the nano-vaccine was 260 nm.

[0319] (6) The nano-vaccine is used for the prevention of type I diabetes

[0320] Same as Example 1.

[0321] (7) Experimental results

[0322] As Figure 13 shown, compared with the mice in the PBS control group, the proportion of mice with type I diabetes treated with the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with whole cell antigen was significantly reduced. Moreover, the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles loaded with KALA polypeptide had a better effect than the nano-vaccine prepared from antigen-presenting cells activated by nanoparticles without KALA polypeptide. Thus, it can be seen that the nano-vaccine described in the present invention has a preventive effect on type I diabetes, and adding KALA polypeptide to the nanoparticles or microparticles that activate antigen-presenting cells helps to improve the efficacy of the nano-vaccine prepared from the activated antigen-presenting cells. In this example, KALA polypeptide was added, and in actual use, one or more substances with the function of increasing lysosomal escape, such as arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH4HCO3, positively charged polypeptides, protamine, histone, etc., can also be loaded.

[0323] Example 13 Nano-vaccine for Preventing Autoimmune Diseases

[0324] (1) Lysis of β cells

[0325] Double the glucose content in the high-glucose medium, and simultaneously add 0.05 μM / L thapsigargin to the medium and incubate NIT-1 cells for 12 hours. Increasing the glucose content and adding thapsigargin can increase the antigen content in β cells. Then, collect the cultured NIT-1 cells, remove the medium by centrifugation, resuspend and lyse the NIT-1 cells with 10% sodium deoxycholate aqueous solution (containing 8 M arginine), and dissolve the lysate components with 10% sodium deoxycholate aqueous solution (containing 8 M arginine) to obtain the antigen source for preparing nanoparticles that activate antigen-presenting cells.

[0326] (2) Preparation of nanoparticles

[0327] In this example, the nanoparticles are prepared by the solvent evaporation method. The PLGA with a molecular weight of 38 KDa - 54 KDa is used as the nanoparticle preparation material. The immunosuppressants used are rapamycin and mycophenolate mofetil. The substances used to increase lysosomal escape are polyarginine and RALA polypeptide. The immunosuppressants, polyarginine, and RALA polypeptide are all loaded inside the nanoparticles. The preparation method is as described above. During the preparation process, the internal lysis components, immunosuppressants, polyarginine, and RALA polypeptide are first lysed inside the nanoparticles by the double emulsion method. Then, 100 mg of PLGA nanoparticles are centrifuged at 13,000 g for 20 min, and the precipitate is resuspended with 4% trehalose and freeze-dried for 48 hours for later use. The average particle size of these nanoparticles is about 260 nm; each 1 mg of PLGA nanoparticles loads about 140 μg of protein or polypeptide components, 0.03 mg of rapamycin and mycophenolate mofetil each, and 0.02 mg of polyarginine and RALA polypeptide each.

[0328] The blank nanoparticle preparation material and preparation method are the same. The average particle size of the blank nanoparticles is about 260 nm. Each 1 mg of PLGA blank nanoparticles loads 0.03 mg of rapamycin and mycophenolate mofetil each, and 0.02 mg of polyarginine and RALA polypeptide each, but does not load any lysis components.

[0329] (3) Activation of antigen-presenting cells

[0330] In this example, DC2.4 cells, B cells derived from splenocytes, and BMDMs were used as mixed antigen-presenting cells, and the preparation methods of the three antigen-presenting cells were the same as above. The DC2.4 cells, B cells, and BMDMs were mixed at a quantity ratio of 1:1:1. 1 mg of nanoparticles loaded with whole-cell antigen was co-incubated with 30 million mixed antigen-presenting cells (10 million DCs + 10 million B cells + 10 million BMDMs) in 25 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2). The incubation system contained IL-7 (100 U / mL), IL-10 (1000 U / mL), IL-4 (500 U / mL), and TGF-β (1000 U / mL). After incubation, the cell precipitate was collected by centrifugation at 400 g for 5 minutes, and the mixed antigen-presenting cells were collected after centrifugal washing twice with PBS for standby. The activated mixed antigen-presenting cells were used as live cell vaccine 3.

[0331] (4) Preparation of nano-vaccine

[0332] Double the glucose content in the high-glucose medium, and simultaneously add 0.05 μM / L thapsigargin to the medium and incubate NIT-1 cells for 18 hours. Then, collect the cultured NIT-1 cells. Mix 30 million NIT-1 cells with 30 million activated mixed antigen-presenting cells from step (3) (10 million DC + 10 million B cells + 10 million BMDM), then wash the mixed cells twice with 4°C phosphate-buffered saline (PBS) containing protease inhibitors. Resuspend the cells in PBS water and sonicate at 4°C with low power (10 W) for 20 minutes. Then centrifuge the sample at 3000 g for 15 minutes and collect the supernatant. Filter the supernatant successively through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 2 μm, 1 μm, 0.45 μm, and 0.22 μm, collect the filtrate, then centrifuge the filtrate at 18000 g for 50 minutes, discard the supernatant, and collect the precipitate. Resuspend the precipitate in PBS to obtain the mixed cell membrane fraction. Then mix the mixed cell membrane fraction with the nanoparticles loaded with whole cell components prepared in step (2) (100 mg) or blank nanoparticles (100 mg) and stir at 1500 RPM for 5 minutes. Then co-incubate at room temperature for 15 minutes, filter and extrude through a 0.45 μm filter membrane, then centrifuge at 13000 g for 25 minutes and resuspend in 10 mL of cryoprotectant aqueous solution (containing 2% trehalose + 2% mannitol + 1% sucrose), and then freeze-dry for 48 hours to obtain the nano-vaccine. Among them, the nano-vaccine obtained by the co-action of the nanoparticles loaded with whole cell components and the mixed cell membrane fraction is nano-vaccine 1, and the particle size of nano-vaccine 1 is 270 nanometers; the nano-vaccine obtained by the co-action of blank nanoparticles and the mixed cell membrane fraction is nano-vaccine 2, and the particle size is 270 nanometers.

[0333] (5) The nano-vaccine is used for the prevention of type I diabetes

[0334] The control group in this study is the PBS group. Female NOD mice at 3 weeks of age were selected for this experiment. In the experiment, there were 10 NOD mice in each group. Starting from the third week, 100 μg of nano-vaccine 1, or 100 μg of nano-vaccine 2, or 3 million mixed antigen-presenting cell vaccines 3 (1 million DC + 1 million B cells + 1 million BMDM), or 100 μL of PBS were subcutaneously injected every 7 days for 6 consecutive weeks. The blood glucose of each group of mice was recorded every day starting from the 8th week. When the blood glucose was higher than 11.0 mmol·L-1, diabetes began to develop. Record the incidence of diabetes in NOD mice at different time periods.

[0335] (6) Analysis of antigen-specific regulatory T cells (T reg )

[0336] Female C57BL / 6 mice at 6 - 8 weeks old were selected as model mice. On day 0, day 7, day 14, day 28, and day 42, each mouse was subcutaneously injected with 100 μg of nano - vaccine 1, or nano - vaccine 2, or 3 million mixed antigen - presenting cell vaccines 3 (1 million DCs + 1 million B cells + 1 million BMDMs), or PBS. The mice were sacrificed on day 45, and the spleens were removed and single - cell suspensions of spleen cells were prepared. B cells and T cells were sorted from mouse spleen cells using magnetic bead sorting. 100 μg of nanoparticles loaded with β - cell whole - cell antigen, 5 million B cells, and 1 million T cells were co - incubated in 5 mL of complete RPMI1640 medium for 48 hours (37 °C, 5% CO2). Then the incubated cells were collected and labeled with live - dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody, and FOXP3 antibody, and then the subsets of T cells were analyzed by flow cytometry for the proportion of CD4 + CD25 + FOXP3 + The proportion of CD4 + T cells among CD4 + The proportion of CD8 + Ly49 + T cells among CD8

[0337] (7) Experimental results

[0338] As Figure 14 shown in a, compared with the PBS group, the proportion of mice with type I diabetes in the vaccine - treated groups was significantly reduced. Moreover, the effect of nano - vaccine 1 was significantly better than that of nano - vaccine 2 and live - cell vaccine 3. This indicates that the nano - vaccine with whole - cell antigen loaded internally and a mixture of cell membranes containing antigens and activated antigen - presenting cells loaded on the surface is better than the nano - vaccine without internal antigen loading but only with a mixture of cell membranes loaded on the surface. It also shows that the nano - vaccine with only immunosuppressants and lysosome - escape substances loaded internally and a mixture of cell membranes containing antigens and activated antigen - presenting cells loaded on the surface is also better than the live - cell vaccine of mixed antigen - presenting cells activated by nanoparticles loaded with whole - cell antigen. In summary, it shows that the nano - vaccine of the present invention with whole - cell antigen loaded internally and a mixture of cell membranes containing mixed cell membranes on the surface has a good preventive effect on type I diabetes.

[0339] As Figure 14 shown in b and 14c, the amount of autoantigen - specific T reg induced by nano - vaccine 1 is more than that of nano - vaccine 2 and nano - vaccine 3. This indicates that loading whole - cell antigen internally and a mixture of cell membranes containing mixed cells on the surface is beneficial for inducing more autoantigen - specific T reg . Because it can inhibit T eff, so the induction can be effective in preventing or treating type I diabetes.

[0340] In this example, the surface of the nano-vaccine is loaded with the membrane components of cells containing self-antigens. In practical applications, the membrane components of extracellular vesicles secreted by cells containing self-antigens can also be used.

[0341] Example 14 Treatment of type I diabetes with nano-vaccine

[0342] (1) Lysis of β cells

[0343] The cultured NIT-1 cells were centrifuged at 400 g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. The obtained cells were inactivated and denatured by ultraviolet light and high-temperature heating respectively, then ultrapure water was added and the cells were repeatedly frozen and thawed 5 times and sonicated to lyse the cells, then nuclease was added and allowed to act for 5 minutes, and then the nuclease was inactivated at 95 °C for 10 minutes. The cell lysate was centrifuged at 5000 g for 10 minutes, and the supernatant was the water-soluble component. The precipitate was dissolved with 10% octyl glucoside to obtain the dissolved original water-insoluble antigen. The water-soluble component and the water-insoluble component were mixed at a mass ratio of 2:1 to obtain the lysate component required for preparing the micro-particles.

[0344] (2) Preparation of the micro-particle system

[0345] In this example, the micro-particle system and the control micro-particles were prepared by the double emulsion method. The molecular weight of the PLGA, the skeleton material of the micro-particles, was 38 KDa - 54 KDa. The immunosuppressant used was methylprednisolone, and the substances used to increase lysosomal escape were NH4HCO3 and melittin. During preparation, the micro-particles internally loaded with the lysate component, methylprednisolone, NH4HCO3 and melittin were first prepared by the double emulsion method, and then 100 mg of the micro-particles were centrifuged at 9000 g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose and dried for 48 h for standby. The average particle size of the micro-particles was about 3.1 μm, and the surface potential was about -7 mV; about 110 μg of protein or polypeptide components were loaded per 1 mg of PLGA micro-particles, containing 0.01 mg of methylprednisolone, 0.02 mg of each of NH4HCO3 and melittin. The preparation materials and methods of the control micro-particles were the same as the above method. The average particle size of the control micro-particles was about 3.1 μm, and the surface potential was about -7 mV; about 110 μg of protein or polypeptide components were loaded per 1 mg of PLGA micro-particles, without loading any immunosuppressant, and only loading 0.02 mg of each of NH4HCO3 and melittin.

[0346] (3) Preparation of antigen-presenting cells

[0347] In this embodiment, the DC2.4 cell line is used as the antigen-presenting cell.

[0348] (4) Activation of antigen-presenting cells

[0349] The micron particles (1000 μg) loaded with whole cancer cell antigens are co-incubated with DC2.4 (10 million) in 15 mL of high-glucose DMEM complete medium for 48 hours (37 °C, 5% CO2); the incubation system contains GM-CSF (2000 U / mL), IL-2 (100 U / mL), IL-10 (2000 U / mL), and IL-13 (200 U / mL).

[0350] (5) Preparation of antigen-presenting cell-derived nano-vaccine

[0351] The incubated DC is collected by centrifugation at 400 g for 5 minutes, and then the cells are washed twice with 4 °C phosphate buffer solution (PBS) containing protease inhibitors. After resuspending the cells in PBS water, they are sonicated at 4 °C with low power (20 W) for 2 minutes. Then the sample is centrifuged at 3000 g for 15 minutes and the supernatant is collected. The supernatant is filtered successively through membranes with pore sizes of 30 μm, 10 μm, 5 μm, 1 μm, and 0.45 μm, and then centrifuged at 15000 g for 60 minutes to obtain the nano-vaccine. The average particle size of the nano-vaccine is 250 nanometers.

[0352] (5) Treatment of type I diabetes with nano-vaccine

[0353] Same as Example 7.

[0354] (6) Experimental results

[0355] As Figure 15 shown, compared with the PBS control group, the type I diabetic mice treated with the nano-vaccine prepared from antigen-presenting cells activated by micron particles were improved after treatment. Moreover, the nano-vaccine prepared from antigen-presenting cells activated by micron particles loaded with both immunosuppressants and lysosome escape substances was superior to the nano-vaccine prepared from antigen-presenting cells activated by micron particles loaded only with lysosome escape substances without immunosuppressants. This shows that immunosuppressants can improve the efficiency of antigen-presenting cell activation by micron particles, which is beneficial for the nano-vaccine prepared from antigen-presenting cells to better induce T cells to transform into regulatory T cells with specific immunosuppressive functions.

[0356] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill 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 manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing an autoimmune disease vaccine derived from pre-activated antigen-presenting cells, characterized in that, Comprising the following steps: S1. Co-incubate antigen-presenting cells with a first delivery particle loaded with whole-cell autoimmunity disease antigen to obtain pre-activated antigen-presenting cells; S2. Prepare the cell membrane of the pre-activated antigen-presenting cells into nanovesicles to obtain the autoimmunity disease vaccine; Or load the cell membrane components of the pre-activated antigen-presenting cells and the cell membrane components of cells containing autoimmunity disease antigens onto a second delivery particle loaded with whole-cell autoimmunity disease antigen to obtain the autoimmunity disease vaccine; Wherein, The first delivery particle or the second delivery particle is independently a nanoparticle or a microparticle; The whole-cell autoimmunity disease antigen is prepared by the following steps: Freeze cells or tissues containing autoimmunity disease antigens, add water for freeze-thaw lysis, collect the supernatant and the soluble part in the precipitate after dissolution by a solubilizing agent to obtain the whole-cell autoimmunity disease antigen; or add a solubilizing agent to cells or tissues containing autoimmunity disease antigens for lysis, and collect the soluble part to obtain the whole-cell autoimmunity disease antigen; In step S1, the co-incubation system includes one of the following combinations during co-incubation: (1) GM-CSF, IL-2, IL-7, IL-10 and CD40 antibody; (2) TGF-β, IL-4, IL-7, IL-10; (3) GM-CSF, IL-2, IL-4 and IL-10, or GM-CSF, IL-2, IL-4, IL-10 and PD-L1 antibody; (4) TGF-β, IL-10, IL-4 and PD-L1 antibody; (5) GM-CSF, IL-2, IL-10 and TGF-β; (6) IL-2, IL-10, IL-4 and TGF-β; (7) IL-10, IL-2, IL-4 and IL-13; (8) IL-13, IL-10, IL-4 and TGF-β; (9) GM-CSF, IL-2, IL-10 and IL-13.

2. The preparation method according to claim 1, characterized in that: The solubilizing agent is selected from one or more of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, proteolytic enzyme, albumin, lecithin, inorganic salt, Triton, Tween, amino acid, glycoside and choline.

3. The preparation method according to claim 1, characterized in that: In step S1, the co-incubation system contains glucose and / or thapsigargin during co-incubation.

4. The preparation method according to claim 1, characterized in that: The antigen-presenting cells are selected from at least one of dendritic cells, B cells and macrophages.

5. The preparation method according to claim 1, characterized in that: The first delivery particle or the second delivery particle is loaded with an immunosuppressive substance; the immunosuppressive substance is selected from one or more of mRNA, DNA, glucocorticoid drugs, calcineurin inhibitors, antimetabolites, antibodies, cytokines, alkylating agents, cyclosporine, rapamycin, tacrolimus, gusperimus, fingolimod, methylprednisolone, tripterygium wilfordii, mycophenolate mofetil, cyclophosphamide, azathioprine, everolimus, sandimmun, cyclosporine polypeptide A, new demethylated cyclosporine, TGF-β, interleukin, ginseng and astragalus membranaceus.

6. The preparation method according to claim 1, characterized in that: One or more of KALA polypeptide, RALA polypeptide, melittin, arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH4HCO3, protamine and histone are loaded on the first delivery particle or the second delivery particle.

7. An autoimmune disease vaccine prepared by the preparation method according to any one of claims 1-6.

8. Use of the autoimmune disease vaccine according to claim 7 in the preparation of a medicament for treating or preventing autoimmune diseases.

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