CN114931633A

CN114931633A - Preparation method and application of autoimmune disease vaccine derived from pre-activated antigen presenting cells

Info

Publication number: CN114931633A
Application number: CN202210654017.3A
Authority: CN
Inventors: 刘密
Original assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-08-23
Also published as: WO2023236331A1

Abstract

The invention relates to a preparation method and application of an autoimmune disease vaccine from a pre-activated antigen presenting cell, wherein the preparation method comprises the following steps: incubating the antigen-presenting cell with the first delivery particle loaded with the autoimmune disease whole cell antigen to obtain a pre-activated antigen-presenting cell; and preparing the pre-activated antigen presenting cell membrane into nano-vesicles or nano-particles or micro-particles with cell membrane components loaded on the surfaces of the particles to obtain the autoimmune disease vaccine. The invention realizes that the vaccine derived from the dendritic cells loads broad-spectrum and diverse cancer cell antigens, simultaneously overcomes the difficult problems of the live cell vaccine such as difficult maintenance of the activity of the dendritic cells and incapability of freeze-drying and long-term storage, and can prepare the vaccine loading broad-spectrum autoimmune disease epitope for preventing and treating autoimmune diseases.

Description

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

Technical Field

The invention relates to the field of immunotherapy, in particular to a preparation method and application of an autoimmune disease vaccine derived from a pre-activated antigen presenting cell.

Background

Autoimmune diseases are a serious threat to human health, and the pathogenesis of the autoimmune diseases is that the human immune system mistakenly recognizes the self substance as a foreign antigen for some reasons and initiates an attack on cells or tissues containing the antigen, resulting in a series of consequences and diseases. In the case of type I diabetes, for some reasons that are not fully studied, the body's immune system mistakenly mistakens certain components in the islets or beta cells for foreign antigens, thereby initiating attack on the beta cells and, in turn, killing most of the beta cells. Since beta cells are the substance of insulin secreted by the human body and insulin is the key substance for controlling blood sugar, when most of the beta cells are killed, the human body cannot generate enough insulin to control blood sugar, so that type I diabetes occurs. Therefore, if the attack of the human immune system on beta cells can be prevented or relieved, the type I diabetes can be prevented or reversed. The vaccine capable of inducing the body to generate immune tolerance is one of the main methods for realizing the aim. Since beta cells are mainly composed of effector T cells (T) _eff ) Aggressive, so inducing recognition of the same antigen as but on effector T cells (T) _eff ) Has the function of inhibitingRegulatory T cells (T) for use _reg ) It is of vital importance. Therefore, the present invention aims to find a method for inducing the generation of regulatory T cells with high efficiency, thereby finding a method capable of effectively preventing and treating autoimmune diseases.

Disclosure of Invention

In order to solve the technical problems, the invention provides an antigen-presenting cell which is derived from nano-particles and/or micro-particles, and a nano-vaccine (NP) or a micro-vaccine (MP) is prepared by using a membrane component of the antigen-presenting cell, and can be used for preventing or treating autoimmune diseases.

The invention provides a preparation method of an autoimmune disease vaccine derived from a pre-activated antigen presenting cell, which comprises the following steps:

s1, co-incubating the antigen presenting cells and the first delivery particles loaded with the autoimmune disease whole cell antigens to obtain pre-activated antigen presenting cells;

s2, preparing the cell membrane of the pre-activated antigen presenting cell into a nano vesicle to obtain the autoimmune disease vaccine;

or loading the cell membrane component of the pre-activated antigen presenting cell on the second delivery particle loaded with the autoimmune disease whole cell antigen to obtain the autoimmune disease vaccine;

wherein, the first and the second end of the pipe are connected with each other,

the first delivery particle or the second delivery particle is independently a nanoparticle or a microparticle;

the autoimmune disease whole cell antigen is prepared by the following steps: freezing cells or tissues containing the autoimmune disease antigen, adding water for freeze-thaw lysis, collecting supernatant and precipitates, dissolving by a dissolving agent and converting into soluble parts to obtain the autoimmune disease whole cell antigen; or adding a lytic agent into cells or tissues containing the autoimmune disease antigen for cracking, and collecting soluble parts to obtain the autoimmune disease whole cell antigen.

Further, the solubilizing agent is selected from one or more of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salts, Triton, tween, amino acids, glycosides and choline.

Further, loading the cell membrane fraction of the pre-activated antigen-presenting cells onto the second delivery particle loaded with the autoimmune disease whole cell antigen, and loading the cell membrane fraction containing the autoimmune disease antigen cells onto the second delivery particle. Wherein the cell membrane fraction comprises cell membranes and/or extracellular vesicle membranes.

Further, when the delivery particle (hereinafter, "delivery particle" means the first delivery particle or the second delivery particle) is loaded with both the membrane component of the antigen-presenting cell and the membrane component of the cell containing the autoimmune disease antigen (hereinafter, each will be described by taking the membrane component of the β cell as an example), the specific steps include:

(1) incubating one or more antigen-presenting cells with the nanoparticles and/or microparticles loaded with the autoimmune disease whole cell antigen for a time to activate the antigen-presenting cells;

(2) mechanically destroying, membrane filtering, gradient centrifuging or chemically treating the activated antigen-presenting cells to obtain cell membrane fragments and/or nano vesicles from the antigen-presenting cells;

(3) subjecting cells (such as beta cells) containing autoimmune disease antigens to mechanical disruption, membrane filtration, gradient centrifugation or chemical treatment to obtain cell membrane fragments and/or nanovesicles derived from the beta cells;

(4) and (3) enabling the products obtained in the steps (2) and (3) to act with the second delivery particles loaded with the autoimmune disease whole cell antigen to obtain the self-immune disease whole cell antigen.

Further, the mechanical disruption means is selected from one or more of ultrasound, homogenization, high-speed stirring, high-pressure disruption, high-shear disruption, swelling, and shrinkage.

Further, the mode of co-action is selected from one or more of co-incubation, sonication, co-extrusion, ultrafiltration, dialysis, stirring, homogenization and homogenization.

Further, in step S1, the incubation system contains cytokines and/or antibodies during co-incubation; the cytokine is selected from one or more of interleukin 1 receptor antagonist, growth factor, interleukin, interferon, tumor necrosis factor, colony stimulating factor, activin and inhibin; the antibody is selected from one or more of a PD1 antibody, a PD-L1 antibody, a CTAL-4 antibody, a TIGIT antibody, a TIM-3 antibody, a LAG-3 antibody, an alpha CD-8 antibody, an alpha CD-28 antibody, an alpha CD-40 antibody, an alpha OX-40 antibody, and an alpha OX-40L antibody.

Further, cytokines include, but are not limited to, interleukin 1 receptor antagonist (IL-1ra), interleukin 2(IL-2), transforming growth factor-beta (TGF-beta), 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), gamma interferon (IFN-gamma), TNF-alpha, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), activin (activins), Inhibins (inhibins).

Preferably, one of the following combinations is included in the incubation system:

(1) GM-CSF, IL-2, IL-7, IL-10, and CD40 antibodies;

(2) TGF-. beta.s, IL-4, IL-7, IL-10 (most preferably);

(3) GM-CSF, IL-2, IL-4, IL-10, and PD-L1 antibodies (PD-L1 antibody is optional);

(4) TGF-beta, IL-10, IL-4, and PD-L1 antibodies;

(5) GM-CSF, IL-2, IL-10, and TGF- β;

(6) IL-2, IL-10, IL-4 and TGF-beta;

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

(8) IL-13, IL-10, IL-4 and TGF-beta;

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

Further, in step S1, the incubation system contains glucose and/or thapsigargin during co-incubation.

Further, the Antigen Presenting Cell (APC) is selected from at least one of a Dendritic Cell (DC), a B cell and a macrophage.

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 mellitus, 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, thyroid autoimmune disease.

The nano vaccine or the micro vaccine is characterized in that: when the autoimmune disease is type I diabetes, the cells containing the antigens related to the type I diabetes are beta cells; the tissue is a tissue containing beta cells, such as pancreatic tissue and/or pancreatic islet tissue.

Furthermore, the activated antigen-presenting cells can be washed appropriately before preparing the nano-vaccine, and the washing solution used in the washing process contains protease inhibitors.

Further, the process of sonicating antigen presenting cells to prepare nano-or micro-vaccines is low power ultrasound (less than 500W); the gradient centrifugation for preparing the nano vaccine or the micron vaccine is the gradient centrifugation with the centrifugal speed increased in sequence; the aperture of the filter membrane used for preparing the nano vaccine or the micron vaccine is sequentially from large to small; the co-action time is greater than 20 seconds.

Further, the particle size of the nano vaccine is more than 30nm and less than 1000 nm; the particle size of the micro vaccine is more than 1 μm and less than 50 μm.

Further, the nanoparticle size for activating antigen-presenting cells ranges from 10nm to 1000nm, and the microparticle size for activating antigen-presenting cells ranges from 1 μm to 50 μm.

Further, the interior and/or surface of the first delivery particle or the second delivery particle is loaded with an immunosuppressive substance.

Further, the substance for suppressing immunity is selected from one or more of microbe-derived immunosuppressant, product of human or animal immune system, mRNA, DNA, inherent immunosuppressant, adaptive immunosuppressant, chemically synthesized drug, fungal polysaccharide and Chinese medicinal material; can also be selected from one or more of glucocorticoid medicine, calcineurin inhibitor, antimetabolite, antibody, cytokine, alkylating agent, plant medicine component and mineral medicine component; can also be selected from one or more of cyclosporine, rapamycin, tacrolimus, guanolimus, fingolimod, methylprednisolone, tripterygium wilfordii, mycophenolate mofetil, cyclophosphamide, azathioprine, everolimus, domethamine, seipine, cyclosporine A, cyclosporine, xindiguanine, anti-IL-2 receptor monoclonal antibody, TGF-beta, interleukin, ginseng and astragalus.

Further, the first delivery particle or the second delivery particle is loaded with a positively charged polypeptide (such as KALA polypeptide, RALA polypeptide, melittin, etc.), arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH ₄ HCO ₃ One or more of protamine and histone.

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

The invention also provides application of the autoimmune disease vaccine in preparation of a medicament for treating or preventing autoimmune diseases.

Further, the antigen presenting cells are derived from one or more of autologous, allogeneic, cell-line, or stem cell differentiation.

Further, the means for loading the whole cell antigen on the surface of the nanoparticle or microparticle comprises at least one of adsorption, covalent attachment, charge interaction, hydrophobic interaction, one or more steps of immobilization, mineralization and encapsulation.

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

Further, the organic synthetic polymer materials include, but are not limited to, polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), Polyglycol (PEG), Polycaprolactone (PCL), Poloxamer, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), Polyethyleneimine (PEI), polytrimethylene carbonate (PTMC), polyanhydride, PDON, PPDO, Polymethylmethacrylate (PMMA), polyamino acid, synthetic polypeptide, etc.; the natural polymer material comprises lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, saccharide, polypeptide, etc.; the inorganic material includes ferric oxide, ferroferric oxide, carbonate, phosphate, etc.

Furthermore, the nanoparticles or microparticles may not be modified during the preparation process, and suitable modification techniques may also be employed to increase the antigen loading of the nanoparticles or microparticles. Modification techniques include, but are not limited to, biomineralization (e.g., silicidation, calcification, magnesiation), gelation, crosslinking, chemical modification, addition of charged species, and the like.

Further, the antigen can be loaded on the surface of the nanoparticle or microparticle by means including, but not limited to, adsorption, covalent attachment, charge interaction (e.g., addition of positively charged species, addition of negatively charged species), hydrophobic interaction, one or more steps of immobilization, mineralization, encapsulation, and the like.

Furthermore, one or more layers of water-soluble antigens and/or water-insoluble antigens loaded on the surfaces of the nano particles or the micro particles are loaded, and when a plurality of layers of water-soluble antigens and/or water-insoluble antigens are loaded on the surfaces, modifiers are arranged between the layers.

Further, the particle size of the particles for activating the antigen-presenting cells is in the order of nanometers or micrometers, which ensures that the particles are phagocytosed by the antigen-presenting cells, and is within a suitable range for improving phagocytosis efficiency. The size of the particle size of the nanoparticles is 1nm-1000nm, more preferably, the size of the particle size is 30nm-1000nm, and most preferably, the size of the particle size is 50nm-600 nm; the microparticles have a particle size of 1 μm to 1000 μm, more preferably a particle size of 1 μm to 100 μm, more preferably a particle size of 1 μm to 10 μm, most preferably a particle size of 1 μm to 5 μm.

Dendritic cell vaccine is a kind of vaccine, and because DC belongs to living cell preparation, there are many defects in the storage, transportation and administration process of activated DC used as vaccine. According to the invention, the antigen presenting cells are activated by using the nano particles and/or the micro particles loaded with the whole cell antigens of the cells or tissues containing the autoimmune disease antigens, so that the antigen presenting cells are loaded with the whole cell antigens with broad spectrum, and then the antigen presenting cells are processed by the methods of mechanical destruction, gradient centrifugation and/or membrane filtration and/or coaction with the particles to prepare the nano vaccine or the micro vaccine, and the obtained nano vaccine or the micro vaccine is loaded with the antigen epitopes and the antigen presenting cell components of the autoimmune disease, so that the nano vaccine or the micro vaccine is easy to store, transport and use, has a certain homing effect after being injected into a human body, is easy to be phagocytized by the antigen presenting cells, can activate broad-spectrum and diverse specific regulatory T cells, and can inhibit more and better effector T cells in an organism.

By the scheme, the invention at least has the following advantages:

(1) the vaccine may incorporate one or more components of the antigen-presenting cell and an epitope of an autoimmune disease into a nano-or micro-vaccine, which may contain at least one component of the antigen-presenting cell, including DC cells, B cells, or macrophages, and thus a nano-or micro-vaccine may have some of the functions and advantages of one or more of the antigen-presenting cells.

(2) The nano vaccine or the micro vaccine can contain one or more components of antigen presenting cells including DC cells, has the characteristic of homing lymph nodes after being injected into a body, and can better activate antigen-specific inhibitory immune response.

(3) The antigen-presenting cells are activated using the nanoparticles and/or microparticles loaded with the whole-cell antigens of the cells and/or tissues containing the autoimmune disease antigens, and then the antigen-presenting cells are used to prepare the nano-vaccine, so the prepared nano-vaccine can be loaded with all the epitopes loaded by the nanoparticles and/or microparticles for activating the antigen-presenting cells, because the nano-vaccine can activate a wide variety of regulatory antigen-specific T cells (tregs).

(4) The nano-vaccine or the micro-vaccine is derived from antigen presenting cells, and the components are biocompatible and degradable, so that the safety is good.

(5) After the antigen presenting cells with cell activity are processed to prepare the nano vaccine or the micro vaccine without any cell activity, the nano vaccine or the micro vaccine is not as harsh as the conditions required by the live antigen presenting cells for storage, transportation and injection.

(6) The membrane component of the antigen presenting cell is wrapped on the delivery particle, and the membrane component of the cell containing the self antigen is loaded on the surface of the nano particle or the micron particle, so that the capacity of loading broad-spectrum antigen and the capacity of having a cell membrane structure on the surface are endowed to the nano vaccine or the micron vaccine, and the effect that some non-bionic membrane particles can not realize can be realized through the effect of loading the bionic membrane on the surface.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present invention may be more readily and clearly understood, reference will now be made in detail to the present invention, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a schematic diagram of the preparation process and application of the nano-or micro-vaccine of the present invention; wherein a is a schematic diagram of collecting water-soluble components and water-insoluble components in a whole cell antigen after lysing cells or tissues containing the autoimmune disease antigen, and then preparing nanoparticles or microparticles; b is a schematic diagram of adopting a dissolving solution containing a dissolving agent to dissolve and lyse a cell whole-cell antigen of a cell or a tissue containing the autoimmune disease antigen and prepare a nano particle or a micro particle; c is a schematic diagram of using the particles prepared in the step a or the step b to activate antigen presenting cells, preparing the antigen presenting cells into nano vaccines or micro vaccines, and using the nano vaccines to prevent or treat autoimmune diseases such as type I diabetes mellitus and the like;

FIGS. 2 to 15 are experimental results of the prevention or treatment of autoimmune diseases such as type I diabetes mellitus using the nano-or micro-vaccine according to examples 1 to 14, respectively; in FIGS. 3, 4 and 14, a is a result in the prevention or treatment of autoimmune diseases such as type I diabetes mellitus, and b and c are CD8 activated by nano vaccine analyzed using flow cytometry ⁺ And CD4 ⁺ Regulatory specific T cell accounts for total CD8 ⁺ And CD4 ⁺ Proportion of T cells.

Detailed Description

The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.

The nano-or micro-vaccine for preventing or treating autoimmune diseases such as type I diabetes mellitus comprises the nano-or micro-vaccine prepared by antigen presenting cells activated by nano-particles and/or micro-particles of cells loaded with autoimmune disease antigens and/or tumor tissue whole cell antigens. The nanoparticles and/or microparticles are loaded with cellular and/or tissue whole cell antigens or mixtures thereof of the autoimmune disease antigens. The preparation process and the application field of the nano-vaccine or the micro-vaccine for preventing or treating the autoimmune diseases such as the type I diabetes and the like are shown in figure 1.

When preparing nanoparticles or microparticles loaded with cell containing autoimmune disease antigen and/or tumor tissue whole cell antigen for activating antigen presenting cells, the cells or tissues can be cleaved, then water-soluble antigen and water-insoluble antigen are respectively collected, and a nano-particle system or a microparticle system is respectively prepared; or directly using a dissolving solution containing a dissolving agent to directly crack cells or tissues and dissolve cell whole cell antigens and prepare a nano-particle system or a micro-particle system. The cell whole cell antigen can be subjected to treatments including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment and the like before or (and) after lysis, and then nano particles or micro particles are prepared; the nano-particles or the micro-particles can also be directly prepared before cell lysis or (and) after cell lysis without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification and nuclease treatment. In some embodiments of the present invention, the tissue cells are inactivated or (and) denatured before being lysed, or inactivated or (and) denatured after being lysed during the actual use process; in some embodiments of the present invention, the inactivation or (and) denaturation treatment before or (and) after cell lysis is uv irradiation and high temperature heating, and during 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. may also be used. Those skilled in the art can understand that in the practical application process, the technical personnel can make appropriate adjustment according to specific situations.

In the in vitro activation of antigen-presenting cells using nanoparticles or microparticles, cytokines and/or antibodies may be used to help increase the efficiency of activation, and the antigen-presenting cells may be autologous or allogeneic, or derived from cell lines or stem cells. The antigen presenting cell can be DC cell, B cell, macrophage or any mixture of the three, and can also be other cells with antigen presenting function.

After the antigen presenting cells are activated, the antigen presenting cells are prepared into nano vaccines or micro vaccines by using a method of mechanically destroying the cells, and then adopting a centrifugal method and/or a membrane filtration method and/or a method of coaction with nano particles or micro particles and the like. Other methods for preparing the nano-vaccine or the micro-vaccine without cell activity from the live antigen presenting cells can be adopted in the actual preparation.

In some embodiments, the specific method for preparing the antigen-presenting cells into the nano-or micro-vaccine after using the nano-or micro-particle activated antigen-presenting cells loaded with cell and/or tissue whole cell antigens containing the autoimmune disease antigens is as follows:

step 1, adding a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a prepared particle raw material of a second predetermined concentration.

In some embodiments, the aqueous phase solution may contain components of the cell lysate as well as the immunosuppressive agent; the components of the cell lysate are water-soluble antigens or original water-insoluble antigens dissolved in a lytic agent such as urea or guanidine hydrochloride. The aqueous solution contains a concentration of water-soluble antigen or a concentration of original water-insoluble antigen, i.e., a first predetermined concentration, that requires a protein polypeptide concentration of greater than 1ng/mL, sufficient to carry sufficient cellular whole cell antigen to activate the relevant cells. The concentration of the immunosuppressant in the initial aqueous phase is greater than 0.01 ng/mL.

In some embodiments, the aqueous phase solution contains components of the tissue lysate and an immunosuppressive agent; the components of the tissue lysate are water-soluble antigens or original water-insoluble antigens dissolved in a lytic agent such as urea or guanidine hydrochloride. The aqueous solution contains a concentration of water-soluble antigen or a concentration of the original water-insoluble antigen, i.e., the first predetermined concentration, that requires a protein polypeptide concentration level of greater than 0.01ng/mL, sufficient whole cell antigen to be loaded to activate the relevant cells. The concentration of the immunosuppressant in the initial aqueous phase is greater than 0.01 ng/mL.

In some embodiments, the raw material for preparing the particles is polylactic-co-glycolic acid (PLGA) or polylactic acid (PLA), and the organic solvent is dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the starting material for preparing the particles ranges from 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.

In the present invention, PLGA, PLA or modified PLGA, PLA is chosen because the material is a biodegradable material and has been approved by the FDA for use as a pharmaceutical adjuvant. Research shows that PLGA and PLA have certain immunoregulation function, so that the PLGA and PLA are suitable to be used as auxiliary materials in the preparation of nano particles or micro particles. In practical application, suitable materials can be selected according to practical situations.

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 between the first predetermined volume of the aqueous phase and the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1: 10. 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 nanoparticles or microparticles produced during the implementation.

Preferably, when the aqueous phase solution is a lysate component solution, the concentration of the protein and the polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL; when the aqueous phase solution is lysate component/immunosuppressant solution, the concentration of protein and polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of immunoadjuvant is more than 0.01ng/mL, preferably 0.01 mg/mL-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 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.

And 2, carrying out ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or homogenizing treatment or microfluidic treatment on the mixed solution obtained in the step 1. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 5psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 100rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-grade and/or micron-grade particles are subjected to ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment, the size of the prepared micro-nano particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the particle size can be changed when the particles are too large or too small.

And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing a third emulsifier with a preset concentration, and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or microfluidic treatment. Adding the mixture obtained in the step 2 into an emulsifier aqueous solution, and continuing to carry out ultrasonic treatment or stirring for nano-crystallization or micro-crystallization. In the invention, the ultrasonic time is more than 0.1 second, such as 2-200 seconds, the stirring speed is more than 50rpm, such as 50-500 rpm, and the stirring time is more than 1 minute, such as 60-6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 20psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-or micro-scale treatment is carried out by ultrasonic treatment, stirring, homogenizing treatment or micro-fluidic treatment, the size of the prepared nano-or micro-particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the change of the particle size can be brought by the over-large or over-small of the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time.

In some embodiments, the aqueous emulsifier solution is an aqueous poly (ethylene-alt-maleic anhydride) (PEMA) solution or polyvinyl alcohol (PVA), the third predetermined volume is 5mL, 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 and the third predetermined volume is set in the range of 1:1.1 to 1:1000, and preferably may be 2: 5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during the implementation in order to control the size of the nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution and the concentration of the emulsifier aqueous solution are all taken according to the values to obtain the nano-particles or micro-particles with proper size.

And 4, adding the liquid obtained after the treatment in the step 3 into a fourth predetermined volume of emulsifier aqueous solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met.

In this step, the aqueous emulsifier solution is a PEMA or PVA solution or other solution.

The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles or microparticles of a suitable size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5 to 1:2000, preferably 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during the implementation to control the size of the nanoparticles or microparticles.

In the present invention, the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition at the rotating speed of more than 100RPM for more than 1 minute, removing the supernatant, and resuspending the remaining precipitate in a fifth preset volume of aqueous solution with a freeze-drying protective agent at a fifth preset concentration or in a sixth preset volume of PBS (or physiological saline).

In some embodiments of the present invention, the pellet obtained in step 5 is resuspended in a sixth predetermined volume of PBS (or physiological saline) without lyophilization, and the subsequent experiments related to the surface adsorption of cancer cell lysate to nanoparticles or microparticles can be directly performed.

In some embodiments of the present invention, the pellet obtained in step 5 is re-suspended in the aqueous solution containing the lyoprotectant, and then freeze-dried, followed by the subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles.

In the invention, the freeze-drying protective agent is Trehalose (Trehalose).

In the present invention, the fifth predetermined concentration of the lyoprotectant in this step is 4% by mass, and is set so as not to affect the lyophilization effect in the subsequent lyophilization.

And 6, freezing and drying the suspension containing the freeze-drying protective agent obtained in the step 5, and then keeping the freeze-dried substance for later use.

Step 7, directly using the suspension containing the nanoparticles, which is obtained in the step 5 and is resuspended in PBS (or normal saline) in a sixth predetermined volume, or using PBS (or normal saline) in a sixth predetermined volume to resuspend the freeze-dried substance containing the nanoparticles or microparticles and the freeze-drying protective agent obtained in the step 6; or mixing the above sample with a seventh predetermined volume of water-soluble antigen or solubilized antigen not water-soluble before use.

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

In some embodiments, the volume of the resuspended nanoparticle suspension is 1mL compared to the volume containing the water-soluble antigen or solubilized proto-water-insoluble antigen in the cell lysate or tissue lysate when the volume of the resuspended nanoparticle suspension is 10 mL. The volume and the proportion of the two can be adjusted according to the needs when in actual use.

And 8, incubating the antigen presenting cells and the prepared nano particles and/or micro particles for a certain time. The tissues and/or cells from which the nanoparticles and/or microparticles are prepared and the antigen-presenting cells may be autologous or allogeneic.

In step 9, the co-incubated antigen-presenting cells and/or cultured cells containing autoantigens, such as beta cells, are collected, and may or may not be washed and then subjected to sonication, mechanical agitation, homogenization, or the like.

And step 10, carrying out gradient centrifugation and/or membrane filtration and/or combined action with nano particles or micro particles and the like on the mechanically treated antigen presenting cells and/or cell samples containing self antigens to prepare the nano vaccine or the micro vaccine.

In other embodiments, the method of preparing a nano-or micro-vaccine is as follows:

the steps 1-4 are the same as above.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition at the rotating speed of more than 100RPM for more than 1 minute, removing the supernatant, and re-suspending the remaining precipitate in a fifth preset volume of solution containing water-soluble and/or non-water-soluble antigens in the cell whole cell antigens at a fifth preset concentration, or re-suspending the remaining precipitate in a fifth preset volume of solution containing the water-soluble and/or non-water-soluble antigens and the immunosuppressant at a fifth preset concentration.

And 6, centrifuging the mixed solution which is processed in the step 5 and meets the preset stirring condition for more than 1 minute at the rotating speed of more than 100RPM, removing the supernatant, resuspending the remaining precipitate in a sixth preset volume of a solidification treatment reagent or a mineralization treatment reagent, performing centrifugal washing after acting for a certain time, and then adding a seventh preset volume of a substance with positive charge or negative charge and performing action for a certain time.

In some embodiments of the present invention, the pellet obtained in step 6 may be resuspended in the seventh predetermined volume of the charged substance before freeze-drying, and the subsequent experiments related to the surface loading of the cell/tissue lysate on the nanoparticles or microparticles may be directly performed.

In some embodiments of the present invention, the precipitate obtained in step 6 is re-suspended in an aqueous solution containing a desiccation protectant, and then dried in vacuum or freeze-vacuum at room temperature, and then dried, and then the relevant experiments of cell lysate adsorption on the surface of nano-or micro-particles are performed.

In the invention, the freeze-drying protective agent is Trehalose (Trehalose) or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the drying protective agent in this step is set so as not to affect the drying effect in the subsequent drying, because the concentration is 4% by mass.

And 7, drying the suspension containing the drying protective agent obtained in the step 6, and then keeping the dried substance for later use.

8, resuspending the suspension containing the nanoparticles in PBS (or physiological saline) obtained in the step 6 with an eighth predetermined volume or resuspending the dried substance containing the nanoparticles or microparticles and the dry protective agent obtained in the step 7 with PBS (or physiological saline) with an eighth predetermined volume for direct use; or mixed with a ninth predetermined volume of water-soluble antigen or water-insoluble antigen.

In the present invention, the modification and antigen loading steps of steps 5-8 can be repeated multiple times to increase the antigen loading. When a substance having a positive or negative charge is added, a substance having the same charge may be added a plurality of times or a substance having different charges may be added alternately.

In some embodiments, the volume of the resuspended nanoparticle suspension is 10mL, and the volume of the cell lysate or tissue lysate containing water-soluble antigen or originally water-insoluble antigen is 0.1-100 mL. The volume and the proportion of the two can be adjusted according to the needs when in actual use.

And 9, incubating the antigen presenting cells and the prepared nano particles and/or micro particles for a certain time. The tissues and/or cells from which the nanoparticles and/or microparticles are prepared and the antigen-presenting cells may be autologous or allogeneic.

The co-incubated antigen-presenting cells and/or cultured cells containing autoantigens, such as beta cells, are collected, and may or may not be washed and then sonicated, mechanically agitated, homogenized, etc., step 10.

And 11, carrying out gradient centrifugation and/or membrane filtration and/or combined action with nano particles or micro particles and the like on the mechanically treated antigen presenting cells and/or cell samples containing self antigens to prepare the nano vaccine or the micro vaccine.

Example 1 DC cell Membrane-based Nanoprotein for the prevention of type I diabetes

NIT-1 cells are a mouse beta cell model and can be used as islet beta cells. First, NIT-1 cells were lysed to prepare water-soluble and water-insoluble fractions of NIT-1 cells. Then, organic high molecular material PLA is used as a nanoparticle framework material, rapamycin is used as an immunosuppressant, a solvent volatilization method is adopted to prepare nanoparticles loaded with a water-soluble component and a water-insoluble component of beta cells, then the particles are used to activate Dendritic Cells (DC), so that the dendritic cells are activated towards a method for activating regulatory T cells, and then the dendritic cells are subjected to ultrasonic treatment to prepare the nano vaccine for preventing type I diabetes.

(1) Lysis of beta cells and collection of fractions

Culturing NIT-1 beta cells in a high-sugar culture medium, collecting the cultured NIT-1 beta cells, centrifuging, removing the culture medium, resuspending the NIT-1 beta cells with ultrapure water, freezing at-20 ℃ to-273 ℃, adding a certain amount of ultrapure water, repeatedly freezing and thawing for more than 3 times, and carrying out ultrasonic treatment to destroy the lysed cells. After cell lysis, centrifuging the lysate for 5 minutes at 3000g, and taking supernatant fluid, namely a water-soluble component of NIT-1 beta cells which can be dissolved in pure water; the insoluble fraction insoluble in pure water in NIT-1. beta. cells was converted to soluble in an 8M aqueous urea solution by adding 8M urea (containing 500mM sodium chloride) to the resulting precipitate to dissolve the precipitate. The water-soluble component derived from the cell lysate and the original water-insoluble component dissolved in 8M urea are the antigens for preparing the DC-activated nanoparticles.

(2) Preparation of nanoparticles

In the embodiment, a multiple emulsion method in a solvent volatilization method is adopted for preparing the nanoparticles, the molecular weight of polylactic acid (PLA) used as a nanoparticle preparation material is 30KDa, rapamycin is used as an immunosuppressant, rapamycin is loaded in the nanoparticles in preparation, the rapamycin and PLGA are dissolved in an organic phase during preparation, and a PEMA is used as an emulsifier. The preparation method is as described above. The water-soluble component and the water-insoluble component are respectively loaded on different nanoparticles and then mixed for use. In the preparation process, firstly, a whole cell lysate component and an immunosuppressant are loaded inside the nanoparticles by a multiple emulsion method, after the cell lysate component and an adjuvant are loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by 10mL of ultrapure water containing 4% trehalose, and then are freeze-dried for 48 hours, before use, 20mg of the freeze-dried nanoparticles are resuspended in 0.9mLPBS, and then are mixed with 0.1mg of the lysate component (60mg/mL) and are acted for 3 minutes at room temperature, so that the nanoparticles loaded with whole cell antigens inside and outside are obtained. The average particle diameter of the nano particles is 300nm, and the surface potential is about-3 mV. About 130. mu.g of protein or polypeptide component per 1mg of PLA nanoparticle and about 0.05mg of rapamycin per 1mg of PLA nanoparticle. The blank nanoparticle preparation material and preparation method are the same, the particle size is 300nm, and the same amount of immunosuppressant is loaded but no lysate component is loaded.

(3) Preparation of bone marrow-derived dendritic cells (BMDCs)

This example illustrates how to prepare BMDCs by taking dendritic cells prepared from mouse bone marrow cells as an example. Firstly, 1C 57 mouse with age of 6-8 weeks is taken out and killed by dislocation of cervical vertebra, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and tweezers. The two ends of the bone are cut off by scissors, the PBS solution is extracted by a syringe, the needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of red blood cell lysate was added to lyse red blood. Lysis was stopped by adding 3mL of RPMI1640 (10% FBS) medium, centrifugation at 400g for 3min and discarding the supernatant. Cells were plated in 10mm culture dishes using RPMI1640 (10% FBS) medium with recombinant mouse GM-CSF (20ng/mL), 37 degrees, 5% CO ₂ The culture was carried out for 7 days. The flasks were gently shaken on day 3 and supplemented with the same volume of RPMI1640 medium containing GM-CSF (20ng/mL) (10% FBS). On day 6, medium was subjected to half-volume change. On day 7, a small number of suspended and semi-adherent cells were collected and tested by flow assay for CD86 ⁺ CD80 ⁺ The cells were in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

(4) Activation of DC

Loading nanoparticles derived from beta cell whole cell antigen (500. mu.g, wherein the nanoparticle of water-soluble component is loaded at 250. mu.g, minus)250 μ g nanoparticles loaded with water insoluble components) or blank nanoparticles (500 μ g) + free lysate were incubated with BMDC (1000 ten thousand) in 15mL RPMI1640 complete medium for 96 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained a combination of cytokines: granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(200U/mL), IL-4(500U/mL), IL-7(200U/mL), IL-10 (1000U/mL).

(5) Preparation of DC-derived nano-vaccine

The post-incubation DCs were collected by centrifugation at 400g for 5 minutes, followed by washing the cells twice with PBS, resuspending the cells in PBS followed by sonication at 7.5W for 20 minutes. And centrifuging the sample at 2000g for 20 minutes, collecting supernatant, centrifuging the supernatant at 7000g for 20 minutes, collecting supernatant, filtering and extruding the supernatant by using a filter membrane of 0.22 mu m, collecting filtrate, centrifuging the filtrate at 15000g for 120 minutes, collecting the filtrate, discarding supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the DC cell membrane-based nano vaccine with the particle size of 120 nanometers. The nano vaccine prepared by using the DC activated by the nano particles loaded with the whole cell antigen is a nano vaccine 1; the nano vaccine prepared by using the blank nanoparticle and the DC activated by the free lysate is a nano vaccine 2.

Or the activated DC in the step (4) is collected and centrifuged at 400g for 5 minutes, then the cells are washed twice by PBS, and the cells are directly used as the DC vaccine 3 after being resuspended in PBS.

(6) Prevention of type I diabetes in mice by nano-vaccine or DC vaccine

The study control groups were PBS group and blank nanoparticle + cell lysate group. Female NOD mice of 3 weeks of age were selected for this experiment. In the experiment, 10 NOD mice per group were injected subcutaneously with 100 μ g nano-

vaccine

1, 100 μ g nano-vaccine 2, or 500 ten thousand DC vaccine 3 every 7 days starting from the third week for 6 consecutive weeks. The PBS control group was injected subcutaneously with 100. mu.L of PBS every 7 days from the third week for 6 consecutive weeks. Mice blood glucose was recorded daily for each group of mice starting at week 8. Onset of diabetes is determined by blood glucose higher than 11.0 mmol.L-1. And recording the diabetes onset of NOD mice at different time periods.

(7) Results of the experiment

NOD mice are type I diabetes model mice. Approximately 70% -85% of female NOD mice, without prophylactic treatment, develop type I diabetes after 24 weeks. As shown in fig. 2, 70% -80% of mice treated with the nano vaccine 2 prepared from PBS or blank nanoparticles + cell lysate activated antigen presenting cells had diabetes after 25 weeks; approximately 50% of mice treated with live cell DC vaccines loaded with whole cell antigens are afflicted with type I diabetes; only about 40% of mice treated with the nano-vaccine 1 prepared using nanoparticle-activated antigen-presenting cells loaded with beta cell whole cell antigen will suffer from type I diabetes after 25 weeks. The nano vaccine prepared by the antigen presenting cell activated by the nano particle loaded with the beta cell whole cell antigen has good prevention effect on type I diabetes, and the prevention effect is superior to that of the antigen presenting cell live vaccine activated by the nano particle.

Example 2 preparation of Nanoprotein vaccine for type I diabetes by antigen presenting cells

(1) Lysis of beta cells and collection of fractions

Culturing NIT-1 beta cells in a high-sugar culture medium, collecting the cultured NIT-1 beta cells, centrifuging, removing the culture medium, resuspending the NIT-1 beta cells with ultrapure water, freezing at-20 ℃ to-273 ℃, adding a certain amount of ultrapure water, repeatedly freezing and thawing for more than 3 times, and carrying out ultrasonic treatment to destroy the lysed cells. After cell lysis is completed, centrifuging the lysate for 10 minutes at 2000g, and taking supernatant fluid, namely water-soluble components of NIT-1 beta cells which can be dissolved in pure water; the non-water-soluble components insoluble in pure water in NIT-1 beta cells can be converted to be soluble in aqueous solution by adding 10% sodium deoxycholate to the obtained precipitate to dissolve the precipitate. And mixing the obtained water-soluble component derived from the cell lysate and the original water-insoluble component dissolved in the sodium deoxycholate according to the mass ratio of 1:1 to obtain the antigen component for preparing the particles for activating the antigen presenting cells.

(2) Preparation of microparticle systems

In the embodiment, the preparation of the micrometer particles adopts a multiple emulsion method in a solvent volatilization method, the molecular weight of PLGA (polylactic-co-glycolic acid) used as a micrometer particle preparation material is 24KDa-38KDa, the adopted immunosuppressant is a mixed immunosuppressant of rapamycin and tacrolimus, the mixed immunosuppressant is distributed in the micrometer particles, and the rapamycin, the tacrolimus and the PLGA are dissolved in an organic phase together during preparation. The preparation method is as described above, during the preparation process, firstly, the beta cell whole cell lysate component and the immunosuppressant are loaded inside the microparticles by a double emulsion method, after the cell lysate component and the immunosuppressant are loaded inside, 100mg of the microparticles are centrifuged at 7000g for 10 minutes, after supernatant fluid is discarded, the precipitate is resuspended by 10mL of ultrapure water containing 4% trehalose, and then the precipitate is frozen and dried for 48 hours for standby. The average particle size of the obtained microparticles is about 1.5 μm, the surface potential of the micro-vaccine is about-7 mV, about 90 μ g of protein or polypeptide component is loaded on each 1mg of PLGA micro-vaccine, and each 1mg of PLGA micro-vaccine is loaded with 0.025mg of rapamycin and tacrolimus respectively. The particle size of the blank micron particles is about 1.4 mu m, the blank micron particle preparation material and the preparation method are the same, and the same amount of rapamycin and tacrolimus are loaded but no cracked water-soluble component and non-water-soluble component are loaded. The preparation method of the microparticles for loading several type I diabetes antigen polypeptides is the same, the loaded polypeptides with equal mass are Insulin B9-23, Insulin A14-20, IGRP 206-214 and GAD 225-244, the average particle diameter of the particles is about 1.5 μm, the surface potential of the vaccine is about-7 mV, and each 1mg PLGA microparticle is loaded with about 90 μ g of polypeptide component and equal amount of immunosuppressant.

(3) Preparation of antigen-presenting cells

This example illustrates how to prepare BMDCs by taking dendritic cells prepared from mouse bone marrow cells as an example. Firstly, 1C 57 mouse with age of 6-8 weeks is taken out and killed by dislocation of cervical vertebra, tibia and femur of hind leg are taken out by operation and put into PBS, and muscle tissue around the bone is removed by scissors and tweezers. The two ends of the bone are cut off by scissors, the PBS solution is extracted by a syringe, the needles are respectively inserted into the marrow cavity from the two ends of the bone, and the marrow is repeatedly washed into a culture dish. Bone marrow solution was collected, centrifuged at 400g for 3min, and then 1mL of red blood cell lysate was added to lyse red blood. Lysis was stopped by adding 3mL of RPMI1640 (10% FBS) medium, centrifugation at 400g for 3min and discarding the supernatant. The cells were cultured in a 10mm culture dish using RPMI1640 (10% FBS) medium, while maintainingRecombinant mouse GM-CSF (20ng/mL), 37 degrees, 5% CO was added ₂ The culture was carried out for 7 days. The flasks were gently shaken on day 3 and supplemented with the same volume of RPMI1640 medium containing GM-CSF (20ng/mL) (10% FBS). On day 6, medium was subjected to half-exchange treatment. On day 7, a small number of suspended and semi-adherent cells were collected and tested by flow assay for CD86 ⁺ CD80 ⁺ The cells were in CD11c ⁺ The proportion of cells is between 15 and 20 percent, and the BMDCs cultured by induction can be used for the next experiment.

After NOD mice were sacrificed, spleens of mice were harvested, single cell suspensions of mouse splenocytes were prepared, and CD19 was isolated from live splenocytes by magnetic bead sorting (dead cells were labeled with live-dead cell dye to remove dead cells) ⁺ B cells are ready for use.

(4) Activation of antigen presenting cells

Whole cell antigen loaded microparticles (500. mu.g) or polypeptide microparticles (500. mu.g) or empty microparticles (500. mu.g) + free lysate were incubated with BMDCs (500 ten thousand) and B cells (500 ten thousand) in 15mL high glucose DMEM complete medium for 72 hours (37 ℃, 5% CO ₂ ) The incubation system contained GM-CSF (2000U/mL), IL-2(200U/mL), IL-7(1000U/mL), IL-10(2000U/mL), and CD40 antibody (20 ng/mL).

(5) Preparation of nano vaccine based on antigen presenting cell

The incubated antigen presenting cells were harvested by centrifugation at 400g for 5 minutes, followed by washing twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 3 minutes of low power (20W) sonication at 4 ℃. And centrifuging the sample at 3000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, centrifuging at 18000g for 60 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, resuspending the precipitate in PBS, and filtering the sample by using a 0.22 mu m membrane filter to obtain the nano vaccine with the particle size of 120 nanometers.

(6) Nano vaccine for preventing type I diabetes of mice

The same as in example 1.

(7) Antigen-specific regulatory T cells (T) _reg ) Analysis of (2)

Female C57BL/6 at 6-8 weeks was selected as model mice, and 100. mu.g of nano-vaccine or PBS was injected subcutaneously into each mouse on days 0, 7, 14, 28 and 42, respectively. On day 45, mice were sacrificed, spleens were harvested and single cell suspensions of splenocytes were prepared, and B cells and T cells were sorted from mouse splenocytes using magnetic bead sorting. Mu.g of beta cell whole cell antigen loaded microparticles, 500 ten thousand B cells and 100 ten thousand T cells were CO-incubated in 5mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ). The incubated cells were then collected and labeled with live and dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody and FOXP3 antibody, and the T cell subpopulations were then analyzed by flow cytometry for CD4 ⁺ CD25 ⁺ FOXP3 ⁺ T cell occupancy CD4 ⁺ Ratio of T cells and CD8+ Ly49 ⁺ T accounts for CD8 ⁺ Proportion of T cells. The beta cell whole cell antigen loaded by the micron particles can be degraded into antigen epitope after being phagocytized by B cells of the antigen presenting cells and presented on the surface of the antigen presenting cells, and the specific T cells capable of recognizing the beta cell whole cell antigen can be activated and highly express the specific surface marker after recognizing the beta cell whole cell antigen epitope. T cells which highly express specific markers analyzed by flow cytometry are regulatory T cells, namely effector T cells (T cells) capable of inhibiting recognition and killing of beta cells _eff ) T of _reg 。

(8) Results of the experiment

As shown in fig. 3a, 70% -80% of mice treated with nano-vaccine prepared from PBS or empty microparticles plus antigen-presenting cells activated by cell lysate suffered from type I diabetes after 25 weeks; 50% of mice treated with nano-vaccine prepared from antigen-polypeptide-loaded, microparticle-activated antigen-presenting cells had diabetes after 25 weeks. Only about 30% of mice treated with the mini-vaccine prepared with the beta cell whole cell antigen loaded microparticle activated antigen presenting cells had type I diabetes after 25 weeks. In conclusion, the nano vaccine provided by the invention has a prevention effect on type I diabetes. This indicates that the loaded antigen polypeptide microparticles activated DC + B cells present limited kinds of antigen epitopes, so that the preparation thereofBeta cell specific T activated by prepared nano vaccine _reg The cell system contains a small number of T cell clones and can inhibit T _reg Specific T recognizing the same antigen _eff There are fewer varieties. The beta cell antigen presented by the DC + B cell activated by the micron particle loaded with the beta cell whole cell antigen has wider spectrum, so the prepared nano vaccine can activate T _reg The cell clone number is wider, and the cell clone number can be inhibited with T _reg Specific T recognizing the same antigen _eff The variety is also very broad, and the effect of preventing the type I diabetes is better.

FIG. 3B shows CD8 induced by whole cell antigen loaded microparticles activated DC + B cells prepared as nano vaccine ⁺ Ly49 ⁺ T cells and CD4 ⁺ CD25 ⁺ FOXP3 ⁺ Respective T cell occupancy of CD8 ⁺ T cells and CD4 ⁺ The proportion of T cells is obviously higher than the proportion induced by the nano vaccine prepared by the polypeptide-loaded microparticles, the blank microparticles, the free lysate-activated DC and the B cells. Therefore, the nano vaccine prepared by the antigen presenting cell activated by the microparticles loaded with the whole cell antigen can better activate, recognize and inhibit T _eff Beta cell-specific T of (2) _reg A cell.

Example 3 antigen presenting cell derived micro-vaccine for prevention of type I diabetes

This example prepared microparticles from a mixture of mouse islet tissue lysate whole cell fraction and beta cell lysate whole cell fraction, then activated antigen-presenting cells with the microparticles, and then used the antigen-presenting cells to prepare a mini-vaccine.

(1) Islet and beta cell lysis and fraction Collection

Killing a mouse, collecting mouse islet tissues, preparing the islet tissues into a single cell suspension after passing through a cell filter screen, adding a proper amount of pure water, repeatedly freezing and thawing for 5 times with ultrasound to destroy lysed cells, centrifuging a lysate for 3 minutes at a rotating speed of 3000g, taking a supernatant as a water-soluble component, and dissolving a precipitate part by using 8M urea aqueous solution (containing 500mM sodium chloride) to obtain a water-insoluble component dissolved by a dissolving agent.

Culturing NIT-1 cells in a high-glucose DMEM complete medium (glucose content is 4 times of normal content) for 24 hours, adding 10 mu g of thapsigargin during the culture process, collecting the cultured NIT-1 cells, removing the medium after centrifugation, resuspending the NIT-1 cells with ultrapure water, adding a certain amount of ultrapure water, repeatedly freezing and thawing for 5 times, and carrying out ultrasonic treatment to destroy the lysed cells. After the cell lysis was completed, the lysate was centrifuged at 2000g for 10 minutes, and the supernatant was taken as a water-soluble component of NIT-1 cells which can be dissolved in pure water. Dissolving the precipitate with 8M urea water solution (containing 500mM sodium chloride) to obtain water-insoluble component dissolved by the dissolving agent. The high glucose condition and the use of thapsigargin can 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.

Mixing the water-soluble component derived from the beta cell lysate and the water-soluble component derived from the islet tissue lysate according to the mass ratio of 1:1 to obtain a water-soluble component mixture; mixing a water-insoluble component derived from a beta cell lysate and a water-insoluble component derived from an islet tissue lysate according to a mass ratio of 1:1 to obtain a water-insoluble component mixture; mixing the water-soluble component mixture and the water-insoluble component mixture according to the mass ratio of 2:1 to obtain the whole cell component mixture, wherein the whole cell component mixture contains the I type diabetes-related whole cell antigen.

(2) Preparation of microparticle systems

In the embodiment, the preparation of the micron particles adopts a multiple emulsion method in a solvent volatilization method, the molecular weight of PLA which is a material for preparing the micron particles is 40KDa, the adopted immunosuppressant is a mixed immunosuppressant of cyclosporin A and tacrolimus, and the used substance for increasing the escape of lysosomes is NH ₄ HCO ₃ And the mixed immunosuppressant and the lysosome escape increasing substance are distributed in the micrometer particles, and the cyclosporin A, the tacrolimus and the PLA are dissolved in an organic phase together during preparation, and NH is added ₄ HCO ₃ Dissolved in the first aqueous phase together with the lysate components. The preparation method is as described above, and during the preparation process, the whole cells are loaded inside the micrometer particles by the multiple emulsion methodLysate fraction, NH ₄ HCO ₃ And immunosuppressant, then centrifuging 100mg micrometer particles at 6000g for 10 minutes, discarding supernatant, resuspending the precipitate with 10mL ultrapure water containing 4% trehalose, and freeze-drying for 48h for later use; this was resuspended in 9mL PBS before use and then 1mL of the lysate fraction (protein concentration 80mg/mL) was added and allowed to act at room temperature for 10min, resulting in a microparticle system loaded with whole cell lysate fractions both inside and outside. The obtained microparticles have average particle diameter of about 2.55 μm, surface potential of-4 mV, protein or polypeptide component loading of about 130 μ g per 1mg PLA micrometer vaccine, cyclosporin A and tacrolimus loading of 0.025mg per 1mg PLA micrometer vaccine, and NH loading ₄ HCO ₃ It was 0.05 mg.

(3) Preparation of antigen-presenting cells

In this example, DC cells derived from peripheral blood and B cells were used as antigen-presenting cells. Peripheral blood was collected from mice after sacrifice, Peripheral Blood Mononuclear Cells (PBMC) were isolated from mice by gradient centrifugation, and CD19 was then separated from mouse PBMC using flow cytometry ⁺ B cell and CD11c ⁺ And mixing the B cells and the DCs according to the quantity ratio of 1:1 to obtain the mixed antigen presenting cells.

(4) Activation of antigen presenting cells

Whole cell antigen loaded microparticles (500. mu.g) were incubated with mixed antigen presenting cells (1000 million cells in total, 500 million DCs and 500 million B cells) in 15mL high glucose DMEM complete medium for 72 hours (37 ℃, 5% CO2), with cytokine combinations 1 TGF-. beta.s (2000U/mL), IL-4(200U/mL), IL-7(1000U/mL), IL-10 (2000U/mL).

Or, whole-cell antigen-loaded microparticles (500. mu.g) were incubated with mixed antigen-presenting cells (1000 million cells in total, 500 million DCs and 500 million B cells) in 15mL high-glucose DMEM complete medium for 72 hours (37 ℃, 5% CO2), and the incubation system contained the cytokine combinations 2: TNF-a (2000U/mL), IL-12(200U/mL), IL-6(1000U/mL), and IL-15 (2000U/mL).

Alternatively, a mixture of antigen-presenting cells (1000 million cells in total, 500 million DCs and 500 million B cells) was incubated in 15mL high-glucose DMEM complete medium for 72 hours (37 ℃, 5% CO2) with cytokine combinations 1 TGF-. beta.s (2000U/mL), IL-4(200U/mL), IL-7(1000U/mL), and IL-10 (2000U/mL).

(5) Preparation of antigen presenting cell or beta cell membrane based micron vaccine

The activated mixed antigen-presenting cells of step (4) (1000 ten thousand, containing 500 ten thousand DCs and 500 ten thousand B cells) were collected by centrifugation at 300g for 4 minutes, and then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, and the cells were resuspended in PBS water and then sonicated at low power (5W) for 30 minutes. And then filtering the sample through membranes with the pore diameters of 10 microns, 5 microns, 2 microns, 1 micron and 0.45 micron in sequence, collecting filtrate, centrifuging the filtrate at 16000g for 20 minutes, discarding precipitates, collecting supernatant, mixing the supernatant with the filtrate and the micron particles (80mg) prepared in the step (2), carrying out ultrasonic treatment at 20W for 3 minutes, incubating for 15 minutes, filtering and extruding through a filter membrane with the particle diameter of 5 microns, collecting filtrate, centrifuging the filtrate at 8000g for 15 minutes, discarding supernatant, collecting precipitates, and carrying out resuspension on the precipitates in PBS to obtain the micron vaccine. Wherein, the micron vaccine prepared by adding the mixed antigen presenting cells activated by the cytokine combination 1 in the co-incubation process of the micron particles and the mixed antigen presenting cells is the micron vaccine 1, and the particle size is 2.60 mu m; the micron vaccine prepared by adding the mixed antigen presenting cells activated by the cytokine combination 2 in the co-incubation process of the micron particles and the mixed antigen presenting cells is the micron vaccine 2, and the particle size is 2.60 mu m; the micro-vaccine prepared by only incubating the mixed antigen presenting cells and the cytokine combination 1 without adding any micro-particles is the micro-vaccine 3 with the particle size of 2.60 mu m.

Alternatively, cultured NIT-1 cells (1000 ten thousand) were collected, the medium was removed after centrifugation, the cells were washed twice with Phosphate Buffered Saline (PBS) containing a protease inhibitor at 4 ℃ and resuspended in PBS water followed by sonication for 30 minutes at low power (5W). And then filtering the sample through membranes with the pore diameters of 10 microns, 5 microns, 2 microns, 1 micron and 0.45 micron in sequence, collecting filtrate, centrifuging the filtrate for 20 minutes at 16000g, discarding precipitates, collecting supernatant, mixing the supernatant with the micron particles (80mg) prepared in the step (2), carrying out ultrasonic treatment for 3 minutes at 20W, incubating for 15 minutes, filtering and extruding through a filter membrane with the particle diameter of 5 microns, collecting filtrate, centrifuging the filtrate for 15 minutes at 8000g, discarding the supernatant, collecting precipitates, and carrying out resuspension on the precipitates in PBS to obtain the micron vaccine 4 with the particle diameter of 2.60 microns.

Or, the mixed antigen-presenting cells (500 ten thousand, 250 ten thousand DC cells +250 ten thousand B cells) and NIT-1 cells (500 ten thousand) after activation in step (4) are collected and mixed at the same time, and then the cells are washed twice using Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, and the cells are sonicated for 30 minutes at low power (5W) after being resuspended in PBS water. And then filtering the sample through membranes with the pore diameters of 10 microns, 5 microns, 2 microns and 1 micron in sequence, collecting filtrate, centrifuging the filtrate at 16000g for 20 minutes, discarding the precipitate, collecting supernatant, mixing the supernatant with the micron particles (80mg) prepared in the step (2), carrying out ultrasonic treatment at 20W for 3 minutes, incubating for 15 minutes, filtering and extruding through a 5-micron filter membrane, collecting filtrate, centrifuging the filtrate at 8000g for 15 minutes, discarding the supernatant, collecting the precipitate, and resuspending the precipitate in PBS to obtain the micron vaccine 5 with the particle size of 2.60 microns.

(6) Micron vaccine for preventing type I diabetes of mice

The study control group was the PBS group. Female NOD mice of 3 weeks of age were selected for this experiment. In the experiment, 10 NOD mice per group were injected subcutaneously every 7 days starting at week three with 100 μ g of nivadose 1, alternatively 100 μ g of nivadose 2, alternatively 100 μ g of nivadose 3, alternatively 100 μ g of nivadose 4, alternatively 100 μ g of nivadose 5, alternatively 100 μ L of PBS for 6 consecutive weeks. Mice blood glucose was recorded daily for each group of mice starting at week 8. The blood sugar is higher than 11.0 mmol.L ^-1 Onset of diabetes. And recording the diabetes onset of NOD mice at different time periods.

(7) Antigen-specific regulatory T cells (T) _reg ) Analysis of (2)

Selecting female C57BL/6 at 6-8 weeks as model mouse, and injecting 100 μ g micrometer vaccine 1, or micrometer vaccine 2, or micrometer vaccine 3 subcutaneously per mouse on days 0, 7, 14, 28 and 42Vaccine 4 or micron vaccine 5. On day 45, the mice were sacrificed, spleens were removed and single cell suspensions of splenocytes were prepared, and B cells and T cells were sorted from the mouse splenocytes using magnetic bead sorting. Mu.g of beta cell whole cell antigen loaded microparticles, 500 ten thousand B cells and 100 ten thousand T cells were CO-incubated in 5mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ). The incubated cells were then collected and labeled with live and dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody and FOXP3 antibody, and the T cell subpopulations were then analyzed by flow cytometry for CD4 ⁺ CD25 ⁺ FOXP3 ⁺ T cell occupancy CD4 ⁺ Ratio of T cells and CD8+ Ly49 ⁺ T accounts for CD8 ⁺ Proportion of T cells. The beta cell whole cell antigen loaded by the micron particles can be degraded into antigen epitope after being phagocytized by B cells of the antigen presenting cells and presented on the surface of the antigen presenting cells, and the specific T cells capable of recognizing the beta cell whole cell antigen can be activated and highly express the specific surface marker after recognizing the beta cell whole cell antigen epitope. T cells highly expressing specific markers analyzed by flow cytometry are regulatory T cells, i.e., effector T cells (T cells) that can inhibit the recognition and killing of beta cells _eff ) T of (A) _reg 。

(8) Results of the experiment

As shown in fig. 4a, 80% of the PBS control mice had type I diabetes after 25 weeks. The proportion of type I diabetes in each micron vaccine treated mouse was significantly reduced. The results show that the various types of micron vaccines prepared by the embodiment have good prevention effect on type I diabetes. Wherein 40% of mice in the microminiature vaccine 2 treated group had diabetes after 25 weeks; about 30% of mice in the micro vaccine 3 treated group had type I diabetes after 25 weeks; 10% of mice in the group treated with the minivaccine 1 had type I diabetes after 25 weeks; 20% of mice in the micro vaccine 4 treated group had type I diabetes after 25 weeks; the mice in the group treated with the mini vaccine 5 did not develop type I diabetes after 25 weeks. This shows that the mixed antigen presenting cell membrane component activated by the particle loaded with the whole cell antigen is loaded on the surface of the particle, which can obviously improve the effect of the vaccine for preventing type I diabetes; moreover, the cell membrane component of the cell containing the I-type diabetes related antigen loaded on the surface of the particle can also improve the effect of the micron vaccine on preventing the I-type diabetes; moreover, the prevention of type I diabetes is best when the surface of the particles is loaded with a mixture of antigen-presenting cell membrane components and antigen-containing cell membrane components at the same time. The micron vaccine 1 is obviously superior to the micron vaccine 3, which shows that the curative effect of the finally prepared particles can be obviously improved by using the particles loaded with the cell whole cell antigen to activate the antigen presenting cells before the cell membrane components of the antigen presenting cells are loaded on the surfaces of the particles. Furthermore, the advantage of micron vaccine 1 over micron vaccine 2 indicates that the addition of specific cytokines when using particles to activate mixed antigen-presenting cells helps to improve the efficacy of the vaccine prepared from the cell membranes of the antigen-presenting cells. In conclusion, the micron vaccine provided by the invention has a prevention effect on type I diabetes.

As shown in FIGS. 4b and 4c, the autoantigen-specific T induced by the mini-vaccine 1 _reg More than micron vaccine 2 and micron vaccine 3; and autoantigen specific T induced by micron vaccine 5 _reg More than micron vaccine 1. This shows that the cell membrane loaded with the whole cell antigen of the internal load cell and the activated antigen presenting cell on the surface, especially the mixed antigen presenting cell, is beneficial to inducing more autoantigen specific T _reg . Because T having cell killing ability can be inhibited _eff Therefore, the induced could be effective in preventing or treating type I diabetes.

This example uses a micro-vaccine, and a nano-vaccine is also suitable. In this embodiment, type I diabetes in autoimmune diseases is taken as an example, and other autoimmune diseases are also applicable in practice.

In this embodiment, the high glucose condition and the use of thapsigargin are used to increase the amount of antigen synthesized by the cells containing antigens associated with autoimmune diseases, and any other method for increasing the amount of antigen synthesized by the cells, such as increasing the intracellular calcium ion content, can be used in practice.

Example 4 Nanoprotein for prevention of type I diabetes

This example illustrates the use of an antigen presenting cell derived nano-vaccine to prevent autoimmune disease in a mouse type I diabetes model. In this example, pancreatic tissue is first lysed to prepare water-soluble and water-insoluble antigens of the pancreatic tissue; then, a nanoparticle system loaded with water-soluble and water-insoluble antigens of pancreatic tissue is prepared. In this example, the method of silicification and addition of charged substances was used to increase the antigen loading, and only one cycle of mineralization treatment was performed. In this example, the nanoparticles are used to activate antigen-presenting cells, and then the antigen-presenting cells are used to prepare the nano-vaccine.

(1) Lysis of pancreatic tissue and Collection of fractions

Mice were sacrificed and pancreatic tissue was harvested. Cutting pancreatic gland tissue, grinding, adding collagenase, incubating in RPMI1640 culture medium for 30min, preparing single cell suspension by cell filter screen, adding appropriate amount of pure water, repeatedly freezing and thawing for 5 times, and performing ultrasonic treatment to destroy the cells. After the cells are lysed, centrifuging the lysate for 5 minutes at the rotating speed of 5000g, and taking supernatant fluid, namely the water-soluble antigen which can be dissolved in pure water; adding 10% Sodium Dodecyl Sulfate (SDS) aqueous solution into the obtained precipitate to dissolve the precipitate, so as to convert the water-insoluble antigen insoluble in pure water into soluble antigen in 10% SDS aqueous solution, and mixing the water-soluble antigen and the water-insoluble antigen according to the mass ratio of 2:1, thus obtaining the antigen raw material source for preparing the particles.

(2) Preparation of nanoparticles

In the embodiment, the nanoparticles and the blank nanoparticles serving as a reference are prepared by a solvent volatilization method, appropriate modification and improvement are performed, and two modification methods of low-temperature silicification and charged substance addition are adopted in the preparation process of the nanoparticles to improve the antigen loading capacity. The molecular weight of PLA which is a nano particle preparation material is 40KDa, the adopted immunosuppressant is fingolimod, and the fingolimod and the PLA are dissolved in an organic phase. Preparation method As described above, in the preparation process, the antigen and the immunosuppressant are loaded inside the nanoparticles by the double emulsion method, after loading the lysis component inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, then 7mL of PBS is used to resuspend the nanoparticles and mix with 3mL of PBS solution containing cell lysate (60mg/mL), then centrifuged at 10000g for 20 minutes, then 10mL of silicate solution (containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0) is used to resuspend, and fixed at room temperature for 10 minutes, then fixed at-80 ℃ for 24 hours, after centrifugation and washing by ultrapure water, 3mL of PBS containing protamine (5mg/mL) and polylysine (10mg/mL) is used to resuspend and act for 10 minutes, then 10000g is centrifuged for 20 minutes and washing, and 10mL of PBS solution containing lysate (50mg/mL) is used to resuspend and act for 10 minutes, then centrifuging at 10000g for 20 minutes, resuspending with 10mL of ultrapure water containing 4% trehalose, and freeze-drying for 48 h; before the particles are used, the particles are resuspended by 7mL of PBS and then 3mL of lysate components (protein concentration is 50mg/mL) are added and acted for 10min at room temperature, and a nanoparticle system which is loaded with lysate inside and outside and is modified by freezing, silicification and adding a charged substance is obtained. The average particle diameter of the nano particles is about 350nm, and the surface potential of the nano particles is about-3 mV; about 260 μ g of protein or polypeptide component is loaded per 1mg of PLA nanoparticle, and the loading of fingolimod in each 1mg of PLA nanoparticle is 0.03 mg.

The preparation material and the preparation method of the contrast polypeptide nano-particle are the same, four beta cell polypeptide antigens with the same mass and quantity are loaded, and the rest are the same as the nano-particle loaded with the whole cell antigen. The Fingolimod loaded on each 1mgPLA nanoparticle of the polypeptide nanoparticles is 0.03mg, the average particle size is about 350nm, and the surface potential of the nanoparticles is about-3 mV. The loaded polypeptide antigens are Insulin B9-23, Insulin A14-20, IGRP 206-.

The particle size of the blank nanoparticle is about 330nm, the preparation material and the preparation method of the blank nanoparticle are the same, and the blank nanoparticle is loaded with the same amount of adjuvant but not loaded with any cell lysate component.

(3) Preparation of DC

After the mice are sacrificed, the lymph nodes of the mice are picked up, and the lymph nodes of the mice are cut into pieces and then filtered by using a cell screen to prepare a lymph node cell single cell suspension. Flow cytometry was then used to sort CD11c from single cell suspensions of lymph node cells ⁺ Of (3) is performed.

(4) Preparation of bone marrow-derived macrophages (BMDM)

Anaesthetizing C57 mice, dislocating, killing the mice with 75% ethanol, cutting a small opening on the back of the mice with scissors, directly tearing the skin to the joint of the lower leg of the mice with hands, and removing the foot joints and the skin of the mice. The hind limb was removed with scissors along the greater trochanter at the base of the mouse thigh, the muscle tissue was removed, the rat was placed in a 75% ethanol-containing petri dish and soaked for 5min, and the 75% ethanol-containing petri dish was replaced with a new one and transferred to a clean bench. The leg bone soaked in ethanol is transplanted into cold PBS for soaking, and the ethanol on the surfaces of the tibia and the femur is washed off, and the process can be repeated for 3 times. The cleaned femur and tibia are separated, both ends of the femur and tibia are cut off by scissors, the cold induction medium is sucked by a 1mL syringe to blow out bone marrow from the femur and tibia, and the flushing is repeated for 3 times until no obvious red color is seen in the leg bone. Repeatedly blowing the culture medium containing the bone marrow cells by using a 5mL pipette to disperse cell aggregates, sieving the cells by using a 70-micron cell filter, transferring the cells into a 15mL centrifugal tube, centrifuging at 1500rpm/min for 5min, removing the supernatant, adding erythrocyte lysate, resuspending and standing for 5min, centrifuging at 1500rpm/min for 5min, removing the supernatant, resuspending by using a cold prepared bone marrow macrophage induction culture medium (a DMEM high-sugar culture medium containing a 15% L929 culture medium), and plating. Cells are cultured overnight to remove other contaminating cells that adhere faster such as fibroblasts and the like. Collecting non-adherent cells, and seeding into a dish or cell culture plate according to the experimental design. Macrophage colony-stimulating factor (M-CSF) was used at a concentration of 40ng/mL to stimulate differentiation of bone marrow cells to mononuclear macrophages. Culturing for 8 days, and observing the macrophage morphological change under a light microscope. After 8 days the cells were harvested by digestion and incubated with anti-mouse F4/80 and anti-mouse CD11b antibodies at 4 ℃ for 30min in the dark, the proportion of induced successful macrophages was identified using flow cytometry.

(5) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (500. mu.g) or polypeptide nanoparticles (500. mu.g) or blank nanoparticles (500. mu.g) + free lysate were incubated with prepared DCs (500 ten thousand) and BMDM cells (500 ten thousand) in 15mL RPMI1640 complete medium for 24 hours (37 ℃, 5% CO) ₂ ) The incubation system contained granulocyte-macrophage colony stimulating factor (GM-CSF, 2000U/mL), IL-2(200U/mL)IL-4(500U/mL), IL-10(2000U/mL) and PD-L1 antibody (10 ng/mL).

(6) Preparation of antigen presenting cell derived nano vaccine

The incubated DCs and BMDMs were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing phosphatase and protease inhibitors, and after resuspending the cells in PBS water, the antigen-presenting cells were mechanically disrupted at 4 ℃ for 10 minutes using a tissue homogenizer at 1500 rpm. Then filtering the sample by membranes with the aperture of 30 μm,10 μm,5 μm, 2 μm and 0.45 μm in sequence, centrifuging the filtrate for 35 minutes at 14000g, removing supernatant, collecting precipitate, resuspending the precipitate in physiological saline containing 4% mannitol, and freeze-drying to obtain the nano vaccine with the particle size of 260 nm.

(7) Nano vaccine prepared from antigen presenting cell for preventing type I diabetes

The same as in example 1.

(8) Results of the experiment

As shown in fig. 5, 70% -80% of mice treated with nano-vaccine prepared from PBS or empty microparticles plus antigen-presenting cells activated by cell lysate suffered from type I diabetes after 25 weeks; the nano vaccine treated mice prepared from antigen-polypeptide loaded nanoparticle activated antigen presenting cells suffered from diabetes in 60% of the mice after 25 weeks. Only about 30% of mice treated with the nano-vaccine prepared using the nanoparticle-activated antigen-presenting cells loaded with pancreatic tissue whole cell antigen suffered from type I diabetes after 25 weeks. In conclusion, the nano vaccine provided by the invention has a good prevention effect on type I diabetes.

Example 5 antigen presenting cell-based Nanoprotein vaccine for prevention of type I diabetes

In this example, the beta cells were first lysed using 6M guanidine hydrochloride. Then, PLA is used as a nanoparticle framework material, tripterygium wilfordii is used as an immunosuppressant to prepare the nanoparticles loaded with the beta cell whole cell antigen. After the nano particles are used for activating antigen presenting cells, the antigen presenting cell membranes are prepared into nano vaccines for preventing type I diabetes.

(1) Lysis of beta cells

Collecting the cultured NIT-1 cell line, centrifuging for 5 minutes at 400g, removing the supernatant, washing twice with PBS, re-suspending and cracking the NIT-1 cell by 6M guanidine hydrochloride, and cracking the NIT-1 cell whole cell antigen and dissolving in 6M guanidine hydrochloride to obtain the antigen raw material source for preparing the nanoparticle system.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a multiple emulsion method. The molecular weight of PLA which is a nano particle preparation material is 30KDa, and the adopted immunosuppressant is tripterygium wilfordii. As mentioned above, in the preparation process, the NIT-1 cell whole cell antigen and Tripterygium wilfordii are loaded in the nanoparticles by a multiple emulsion method, then 100mg of the nanoparticles are centrifuged at 13000g for 25 minutes, and then 10mL of ultrapure water containing 4% trehalose is used for resuspension and then is frozen and dried for 48 hours for later use. The average particle diameter of the nano particles is about 250nm, and the surface potential of the nano particles is about-4 mV; every 1mg of PLA nano particle is loaded with about 90 mug of protein or polypeptide component and 0.02mg of tripterygium wilfordii. The nano-particle is used as a nano-vaccine 3 in a mouse animal experiment.

(3) Preparation of BMDC

The preparation method is the same as example 1.

(4) Activation of antigen presenting cells

Whole cell antigen loaded microparticles (500. mu.g) were incubated with BMDCs (1000 ten thousand) in 20mL high glucose DMEM complete medium for 72 hours (37 ℃, 5% CO) ₂ ) The incubation system contained TGF-. beta.s (1000U/mL), IL-10(2000U/mL), IL-4(500U/mL) and PD-L1 antibody (10 ng/mL).

(5) Preparation of nano vaccine based on antigen presenting cell membrane

The incubated BMDCs (1000 ten thousand) were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, resuspended in PBS water and then treated with ultrasound (20W) at 4 ℃ for 1 minute, and then homogenized using a high pressure homogenizer (working pressure 5MPa) for 1 minute. And (2) centrifuging the sample at 1000g for 15 minutes, collecting supernatant, centrifuging at 6000g for 15 minutes, discarding the precipitate, collecting supernatant, centrifuging at 16000g for 90 minutes, discarding supernatant, collecting the precipitate, mixing the precipitate with the whole-cell antigen-loaded nanoparticles (50mg) prepared in the step (2) after heavy suspension, performing ultrasonic treatment at 10W for 30 seconds, incubating for 15 minutes, filtering and extruding through a 0.45-micrometer filter membrane, collecting filtrate, centrifuging the filtrate at 12000g for 20 minutes, discarding supernatant, collecting the precipitate, and re-suspending the precipitate in physiological saline to obtain the nano vaccine 1 with the particle size of 260 nm.

The incubated BMDCs (1000 ten thousand) were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, resuspended in PBS water and then treated with ultrasound (20W) at 4 ℃ for 1 minute, and then homogenized using a high pressure homogenizer (working pressure 5MPa) for 1 minute. And then centrifuging the sample at 1000g for 15 minutes and collecting supernatant, then centrifuging at 6000g for 15 minutes and discarding the precipitate, centrifuging at 16000g for 90 minutes and discarding the supernatant and collecting the precipitate, and re-suspending the precipitate by using PBS to obtain the nano vaccine 2 only containing activated DC cell membrane components, wherein the particle size is 130 nm.

(6) Nanometer vaccine for preventing type I diabetes

The control group of the study was the PBS group. Female NOD mice of 3 weeks of age were selected for this experiment. In the experiment, 10 NOD mice per group were injected subcutaneously with 100 μ g Nanoprotein 1, alternatively 100 μ g Nanoprotein 2, alternatively 100 μ g Nanoprotein 3, alternatively 100 μ L PBS, every 7 days from the third week for 6 consecutive weeks. Mice blood glucose was recorded daily for each group of mice starting at week 8. Onset of diabetes is determined by blood glucose higher than 11.0 mmol.L-1. And recording the diabetes onset of NOD mice at different time periods.

(7) Results of the experiment

As shown in fig. 6, 80% of PBS-treated mice had type I diabetes after 25 weeks; 40% of mice treated with nano-vaccine 2 had diabetes after 25 weeks; 40% of mice treated with the nano-vaccine 3 had diabetes after 25 weeks; 20% of the mice treated with the nano-vaccine 1 had diabetes after 25 weeks. This shows that nano vaccine 1 is superior to nano vaccine 2 and nano vaccine 3, and has cancer cell whole cell antigen loaded inside and activated antigen loaded on the surfaceThe cell membrane component of the presenting cell is beneficial to improving the efficacy of the nano vaccine. The nano vaccine 2 is prepared by using a DC cell membrane activated by the nano particle 3, and is a nano particle prepared by a vesicular loaded cell membrane component; the nano vaccine 1 is a nanoparticle with whole cell antigens loaded inside and cell membranes loaded on the surfaces of the solid spheres of the cell membranes presenting the complex antigens on the surfaces, which shows that the solid nano vaccine with the whole cell antigens loaded inside and the membrane components loaded on the surfaces has better effect than the vesicle nano vaccine only with the antigens loaded on the surfaces. Because the surface of the activated antigen presenting cell membrane is provided with a compound formed by combining MHC molecules and antigen polypeptides, the surfaces of the nano vaccine 1 and the nano vaccine 2 are loaded with the compound formed by combining the MHC molecules and the antigen polypeptides. The nano vaccine 1 with the membrane structure can be directly prepared

T cells are induced into regulatory T cells (T) with negative regulatory function _reg ) Or the antigen loaded by the antigen presenting cell after phagocytosis is degraded and presented by the antigen presenting cell to indirectly activate regulatory T cell (T cell) _reg ). In conclusion, the nano vaccine provided by the invention has a good prevention effect on type I diabetes.

Example 6 Nanoprotein for prevention of type I diabetes

In this example, islet tissue was first lysed using 8M urea and the islet tissue lysate components were solubilized. Then, PLA is used as a nanoparticle framework material, cyclophosphamide is used as an immunosuppressant to prepare nanoparticles loaded with a whole-cell antigen, and the nanoparticles are used for activating antigen presenting cells and then utilizing the antigen presenting cells to prepare the nano vaccine for preventing autoimmune diseases, namely type I diabetes.

(1) Lysis of islet tissue

After the mice are sacrificed, islet tissue of the mice is extracted and separated according to the experimental procedure. Islet tissue is ground and passed through a cell strainer, the cells are lysed using an appropriate amount of 8M urea, and the cell lysate is lysed using 8M urea. The above is the source of the antigen raw material for preparing the nanoparticle system.

(2) Preparation of nanoparticle systems

In this example, the nanoparticles were prepared by solvent evaporation. The molecular weight of PLA which is used as a material for preparing the nano particles is 20KDa, the adopted immunosuppressant is cyclophosphamide, and a lysate component and the cyclophosphamide are loaded in the nano particles. The preparation method is as described above, in the preparation process, firstly, a double emulsion method is adopted to load a lysate component and an immunosuppressant in the nano particles, after an antigen lysis component and an immunosuppressant are loaded in the nano particles, 100mg of the nano particles are centrifuged at 12000g for 20 minutes, and after 10mL of ultrapure water containing 4% trehalose is used for resuspension, the nano particles are frozen and dried for 48 hours, so that freeze-dried powder is obtained for later use. The average particle diameter of the nano particles is about 280nm, and the surface potential of the nano particles is about-3 mV; about 100. mu.g of protein or polypeptide component was loaded per 1mg of PLA nanoparticles, and 0.04mg of cyclophosphamide was loaded per 1mg of PLA nanoparticles. The blank nanoparticles are prepared from the same materials and by the same preparation method, the particle size is about 280nm, and the blank nanoparticles are loaded with the same amount of immune preparation but are not loaded with any lysate component. The control nanoparticles are loaded with four antigen polypeptides (Insulin B9-23, Insulin A14-20, IGRP 206-214 and GAD 225-244) with equal mass to replace lysate components, the rest are the same as the nanoparticles loaded with the whole cell antigen, the content of cyclophosphamide loaded on each 1mg of PLA nanoparticles of the control nanoparticles is 0.04mg, the particle size is about 280nm, the surface potential is about-3 mV, and each 1mg of PLA nanoparticles is loaded with about 100 mu g of polypeptide components.

(3) Preparation of DC and B cells

Mouse lymph nodes were harvested after sacrifice to prepare single cell suspensions of mouse lymph nodes, and then CD11c was separated from the single cell suspensions of lymph node cells using flow cytometry ⁺ DC and CD19 ⁺ B cells.

(4) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (500. mu.g) or polypeptide nanoparticles (500. mu.g) were incubated with DCs (500 ten thousand) and B cells (500 ten thousand) in 20mL of high-glucose DMEM complete medium for 72 hours (37 ℃, 5% CO) ₂ ) Or, whole cell antigen-loaded nanoparticles (500. mu.g) were incubated with DCs (1000 ten thousand) in 20mL high-glucose DMEM complete medium for 72 hours (37 ℃, 5%CO ₂ ) (ii) a The incubation system contained GM-CSF (1000U/mL), IL-2(100U/mL), IL-10(2000U/mL), and TGF-. beta.s (2000U/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated DC (500 ten thousand) and B cells (500 ten thousand) were collected by centrifugation at 400g for 5 minutes or only the DC cells (1000 ten thousand) were collected, and then the cells were washed twice with Phosphate Buffered Saline (PBS) at 4 ℃ containing a protease inhibitor, and after resuspension in PBS water, the cells were disrupted at 4 ℃ for 25 minutes with stirring at 2000rpm using a homogenizer. And (3) centrifuging the sample at 3000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, mixing the obtained supernatant with the corresponding nano particles (20mg) for activating the antigen presenting cells prepared in the step (2), stirring at 2000RPM for 2 minutes, filtering and extruding by using a 0.45-micrometer filter membrane, collecting filtrate, centrifuging the filtrate at 12000g for 30 minutes, removing supernatant, collecting precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine, wherein the particle size of the nano vaccine is 300 nanometers.

(6) Nano vaccine for preventing type I diabetes

The same as in example 1.

(7) Results of the experiment

As shown in fig. 7, 70% -80% of mice treated with nano-vaccine prepared from PBS or empty microparticles plus antigen-presenting cells activated by cell lysate suffered from type I diabetes after 25 weeks; the rate of type I diabetes of mice treated by nano vaccine is obviously reduced. In conclusion, the nano vaccine provided by the invention has a good prevention effect on type I diabetes. In addition, the prevention effect of the nano vaccine prepared by DC and B cells activated by nanoparticles loaded with whole cell antigens is superior to that of the nano vaccine prepared by DC and B cells activated by nanoparticles loaded with four antigen polypeptides. Moreover, the effect of the nano vaccine prepared by mixing DC activated by the nano particles loaded with the whole cell antigen and B cells is better than that of the nano vaccine prepared by DC activated by the nano particles loaded with the cancer cell whole cell antigen, which shows that the effect of the nano vaccine prepared by a plurality of antigen presenting cells activated by the nano particles is better. This is probably because a part of the components in activated B cells can help to enhance the activation of specific T cells by nano-vaccines prepared by antigen presenting cells.

EXAMPLE 7 Nanoprotein made by activated antigen presenting cells for the treatment of type I diabetes

This example illustrates the use of nanoparticle-activated antigen-presenting cells to prepare nanocavities and to use such vaccines for the prevention and treatment of autoimmune diseases, using type I diabetes as a model. Beta cells (NIT-1 cells) were first lysed to prepare a whole-cell water-soluble component and a water-insoluble component, and the water-soluble component and the water-insoluble component were mixed at a mass ratio of 1:1. Then, PLA is used as a nanoparticle framework material, rapamycin and guanipimus (Guspeimus) are used as immunosuppressants to prepare nanoparticles, and the nanoparticles are used for preparing a nano vaccine to treat type I diabetes after in vitro activation of antigen presenting cells.

(1) Lysis of beta cells and collection of fractions

After collecting the cell-cultured NIT-1 cell line, the cells were swelled with an appropriate amount of pure water and then freeze-thawed repeatedly 5 times with ultrasound to destroy the lysed cells. After the cells are cracked, centrifuging the lysate for 5 minutes at the rotating speed of more than 5000g and taking supernatant fluid as a water-soluble component which can be dissolved in pure water; the water-insoluble fraction insoluble in pure water can be converted to soluble in an aqueous solution by adding an aqueous solution containing 8M urea and 2% arginine to the resulting precipitate to dissolve the precipitate. Mixing the whole-cell water-soluble component and the water-insoluble component according to the mass ratio of 1:1 to obtain the raw material source for preparing the nano particles.

(2) Preparation of nanoparticles

In this embodiment, the nanoparticles are prepared by a solvent evaporation method. The molecular weight of PLA which is a material for preparing the nano particles is 20KDa, the adopted immunosuppressants are rapamycin and guansirolimus, and the immunosuppressants are distributed in the nano particles. Preparation method As mentioned above, in the preparation process, firstly, the lysate mixture and the immunosuppressant are loaded inside the nanoparticles by using the multiple emulsion method, after the lysate and the adjuvant are loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and are resuspended by using 10mL of ultrapure water containing 4% trehalose, and then are frozen and dried for 48 hours. Before use, 20mg of nanoparticles are resuspended in 0.9mL of PBS and mixed and incubated in 0.1mL of a sample containing a lysate mixture (80mg/mL) for 5 minutes at room temperature. The average particle diameter of the nano particles is about 280nm, and the surface potential of the nano particles is about-3 mV; each 1mg of PLGA nano particle is loaded with about 140 mug of protein or polypeptide component, and each 1mg of PLGA nano particle contains 0.03mg of rapamycin and guansirolimus respectively.

(3) Preparation of antigen-presenting cells

Peripheral blood was collected from mice after sacrifice of C57BL/6, Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD11C was selected from PBMC using flow cytometry ⁺ And (6) DC. Both DC and BMDM were used as antigen presenting cells in this example. BMDM was prepared as in example 4.

(4) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (1000. mu.g) were incubated with peripheral blood-derived DCs (500 ten thousand) and BMDM (500 ten thousand) in 20mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ) Or, the nanoparticles (500. mu.g) loaded with cancer cell whole cell antigens were incubated with DCs (1000 ten thousand) derived from peripheral blood in RPMI1640 complete medium for 72 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (1000U/mL), IL-2(100U/mL), IL-10(2000U/mL), and IL-4 (500U/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated peripheral blood-derived DCs (500 ten thousand) and BMDMs (500 ten thousand) were collected by centrifugation at 400g for 5 minutes or only the peripheral blood-derived DCs (1000 ten thousand) were collected, and then the cells were washed twice with a Phosphate Buffered Saline (PBS) containing a protease inhibitor at 4 ℃, and then resuspended in PBS water and treated in a high-pressure homogenizer (5000bar) for 5 minutes. And (3) centrifuging the sample at 2000g for 15 minutes, collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes, collecting supernatant, incubating the supernatant and the corresponding nanoparticles (60mg) prepared in the step (2) at normal temperature for 1 hour, filtering and extruding the mixture by using a 0.45-micrometer filter membrane, collecting filtrate, centrifuging the mixture at 13000g for 20 minutes, collecting the supernatant, discarding the precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 320 nanometers.

(6) Nanometer vaccine for treating type I diabetes

Female NOD mice, 25 weeks old, that had developed diabetes, were selected for this experiment. In the experiment, 10 NOD mice were per group. Each mouse was injected subcutaneously with 100 μ g of nano-vaccine or PBS every 3 days starting on day 0 for 6 consecutive times. Mice blood glucose was recorded daily for each group of mice. The onset of diabetes is determined by blood sugar higher than 11.0 mmol.L-1, and the remission and cure of diabetes is determined by blood sugar lower than 11.0 mmol.L-1 for three consecutive days. And recording the diabetes onset of NOD mice at different time periods.

(7) Results of the experiment

As shown in fig. 8, the blood glucose in PBS control mice was consistently higher than normal. And the type I diabetes of the mice in the nanometer vaccine treatment group is cured. Moreover, the effect of the nano vaccine prepared by mixing the activated DC and the macrophage is better than that of the nano vaccine prepared by the DC activated by the nano particles, which shows that the effect of the nano vaccine prepared by a plurality of antigen presenting cells activated by the nano particles is better. In conclusion, the nano vaccine prepared by the antigen presenting cell has a good treatment effect on type I diabetes.

EXAMPLE 8 Nanoprotein preparation of activated antigen presenting cells for the treatment of type I diabetes

In this example, type I diabetes is used as a model to illustrate how to use a nano-particle loaded with a beta cell whole cell antigen to activate an antigen presenting cell, and then use a nano-vaccine prepared from the antigen presenting cell to treat type I diabetes.

(1) Lysis of beta cells and Collection of fractions

Collecting cultured NIT-1 cells, using 8M urea aqueous solution (containing 500mM sodium chloride) to crack NIT-1 cells, and using 8M urea aqueous solution to dissolve lysate component to obtain antigen source for preparing nano particles.

(2) Preparation of nanoparticles

In this example, the nanoparticles were prepared by a multiple emulsion method. The molecular weight of PLGA used as a nano particle preparation material is 24-38 KDa, and the adopted inhibitors are rapamycin and everolimus. After loading the lysis fraction and immunosuppressant internally, 100mg of nanoparticles were centrifuged at 10000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and then lyophilized for 48 hours for use. The average particle diameter of the nano particles is about 360nm, and the surface potential of the nano particles is about minus 5 mV; each 1mg of PLGA nanoparticle is loaded with about 80 mug of protein and polypeptide components, and each 1mg of PLGA nanoparticle is loaded with 0.02mg of rapamycin and everolimus respectively.

The preparation material and the preparation method of the blank nano-particle are the same as those of the blank nano-particle, the particle size is about 350nm, and only the same amount of adjuvant is loaded but no lysate component is loaded.

(3) Preparation of DCs

The DC is a mixed DC of a DC derived from peripheral blood and a BMDC. BMDC was prepared as above. Peripheral blood was collected from mice after sacrifice of C57BL/6, Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood, and CD11C was selected from PBMC using flow cytometry ⁺ DC。

(4) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (800. mu.g) or blank nanoparticles (800. mu.g) + equivalent amounts of free lysate were incubated with peripheral blood-derived DCs (800 ten thousand) and BMDCs (800 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) The incubation system contained IL-2(100U/mL), IL-10(2000U/mL), IL-4(1000U/mL) and TGF-. beta.s (1000U/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated peripheral blood-derived DCs (800 ten thousand) and BMDCs (800 ten thousand) were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing a protease inhibitor, and after resuspension in PBS water, treated at 1000rpm for 3 minutes using a homogenizer after 1 minute of low power (20W) sonication at 4 ℃. The sample was then centrifuged at 3000g for 15 minutes and the supernatant collected, which was collected after centrifugation at 8000g for 15 minutes. And (3) carrying out ultrasonic treatment on the supernatant and corresponding nanoparticles (50mg) prepared in the step 2 in the example 1 and the DSPE-PEG-CD32 monoclonal antibody for 2 minutes at 10W, filtering and extruding the mixture by using a filter membrane of 0.45 mu m, collecting filtrate, centrifuging the filtrate at 15000g for 30 minutes, collecting the supernatant, discarding the supernatant, collecting precipitate, and carrying out resuspension on the precipitate in PBS to obtain the nano vaccine 1. Or carrying out ultrasonic treatment on the supernatant and corresponding nano particles (50mg) prepared in the step 2 in the example 1 for 2 minutes at 10W, filtering and extruding by using a filter membrane with the diameter of 0.45 mu m, collecting filtrate, centrifuging the filtrate at 15000g for 30 minutes, collecting the supernatant, discarding the precipitate, and carrying out heavy suspension on the precipitate in PBS to obtain the nano vaccine 2. The particle size of the nano vaccine 1 and the particle size of the nano vaccine 2 are both 380 nanometers.

(6) Nanometer vaccine for treating type I diabetes

The same as in example 7.

(7) Results of the experiment

As shown in fig. 9, blood glucose was consistently higher than normal in the nanobridge-treated mice prepared from PBS control and blank nanoparticle-activated antigen-presenting cells. And partial mice type I diabetes mellitus can be cured by the nano vaccine prepared by the antigen presenting cells activated by the nano particles loaded with the whole cell antigen. Moreover, the effect of the nano-vaccine with the CD32 monoclonal antibody target is better than that of the nano-vaccine without the target. In conclusion, the nano vaccine prepared by the antigen presenting cell has a good treatment effect on type I diabetes. In the nano vaccine of the present embodiment, the CD32 monoclonal antibody is used as the target head for active targeting, and in practical applications, any target head having the ability to target cells, such as mannose, mannan, CD205 monoclonal antibody, CD19 monoclonal antibody, may also be used.

Example 9 antigen presenting cell-based Nanoprotein for the prevention of type I diabetes

This example illustrates how to prepare microparticles loaded with beta cell pancreatic tissue whole cell antigen using mouse pancreatic tissue, and then to prepare antigen-presenting cells into nano-vaccine after activating the antigen-presenting cells using the microparticles, and to use the nano-vaccine to prevent type I diabetes. The pancreas part contains mouse beta cells and can be used as a source of islet beta cells for preparing vaccines. First, pancreatic tissues of mice were harvested and water-soluble components and water-insoluble components of whole cells of the obtained tissues were prepared, respectively. Then, organic polymer material PLGA is used as a nanoparticle framework material, mRNA for encoding TGF-beta and rapamycin are used as immunosuppressants, a solvent volatilization method is adopted to prepare microparticles loaded with whole cell antigens, and the antigen presenting cells activated by the microparticles are used to prepare the nano vaccine.

(1) Collection of pancreatic tissue Whole cell antigens

BALB/c mice were sacrificed and pancreatic tissue was harvested from the mice. Cutting pancreatic tissue into pieces, grinding, adding appropriate amount of pure water through cell filter screen, and repeatedly freezing and thawing and ultrasonic processing for at least 8 times. After the tissue cells are cracked, irradiating the tissue cell lysate for 15 minutes by using ultraviolet rays, then heating the lysate for 10 minutes at 95 ℃, then centrifuging the tissue lysate for 5 minutes at the rotating speed of more than 3000RPM, and taking supernatant fluid, namely water-soluble components which can be dissolved in pure water in the tissue cells; the original water-insoluble component insoluble in pure water can be converted to be soluble in 8M urea aqueous solution by adding 8M urea aqueous solution to the obtained precipitate to dissolve the precipitate. Mixing the water-soluble component and the original water-insoluble component dissolved in 8M urea according to the mass ratio of 1:1 to obtain the antigen source for preparing the microparticles for activating the antigen presenting cells.

(2) Preparation of microparticles for activation of antigen-presenting cells

In the embodiment, the micrometer particles are prepared by a multiple emulsion method in a solvent volatilization method, PLGA used as a micrometer particle preparation material has the molecular weight of 24-38 KDa, the adopted immunosuppressants are mRNA for coding TGF-beta and rapamycin, the mRNA for coding the TGF-beta and the rapamycin are distributed in the micrometer particles, and the mRNA is dissolved in a water phase and the rapamycin is dissolved in an organic phase during preparation. The preparation method is as described above, the average particle diameter of the microparticles is about 1.5 μm, the surface potential Zeta potential is about-8 mV, about 90 μ g of protein or polypeptide component is loaded in each 1mg of PLGA microparticles, and 0.01mg of mRNA is loaded in each 1mg of PLGA microparticles. The blank microparticles, which were around 1.5 μm in size, were loaded with equal amounts of mRNA but not with any lysate fraction.

(3) Preparation of BMDC and B cells

BMDC was prepared as in example 1. The preparation of B cells derived from mouse peripheral blood was performed as described above.

(4) Activation of antigen presenting cells

Incubating microparticles loaded with cellular whole cell antigen (1000 μ g) or blank microparticles (1000 μ g) with BMDCs (1000 ten thousand) in 15mL high glucose DMEM complete medium for 48 hours (37 ℃, 5% CO 2); or whole cell antigen loaded microparticles (1000 μ g) were incubated with B cells (1000 ten thousand) in 15mL high glucose DMEM complete medium for 72 hours (37 ℃, 5% CO 2); the incubation system contained IL-10(2000U/mL), IL-2(500U/mL), IL-4(200U/mL), and IL-13 (200U/mL).

(5) Preparation of nano vaccine based on antigen presenting cell

The incubated DC or B cells were harvested by centrifugation at 400g for 5 minutes, then washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspended in PBS water and sonicated at 4 ℃ for 10 minutes with low power (10W). And filtering the sample through membranes with the pore diameters of 10 micrometers, 5 micrometers, 2 micrometers, 1 micrometer and 0.45 micrometer in sequence, collecting filtrate, centrifuging at 14000g for 40 minutes, removing supernatant, collecting precipitate, and resuspending the precipitate in PBS to obtain the nano vaccine with the particle size of 160 nm.

(6) Nano vaccine for preventing type I diabetes

The same as in example 1.

(7) Results of the experiment

As shown in figure 10, the proportion of type I diabetes in the mice treated with the nano-vaccine prepared from whole-cell antigen-loaded microparticle-activated antigen-presenting cells was significantly reduced compared to the mice treated with the nano-vaccine prepared from PBS and blank microparticle + cell lysate-activated antigen-presenting cells. Moreover, the effect of the nano vaccine prepared by the DC activated by the microparticles is better than that of the nano vaccine prepared by the B cells activated by the microparticles. Therefore, the nano vaccine has a prevention effect on type I diabetes, and the nano vaccines prepared by different antigen presenting cells have different effects.

EXAMPLE 10 Nanoprotein preparation of activated antigen presenting cells for the prevention of type I diabetes

In this example, type I diabetes is used as a model to illustrate how to use a nanoparticle loaded with a beta cell whole cell antigen to activate antigen-presenting cells, and then use a nano-vaccine prepared from the antigen-presenting cells to prevent type I diabetes. Furthermore, appropriate treatment of the beta cells prior to lysis can increase the antigen content and thus increase the efficiency of antigen-loaded particles in activating antigen-presenting cells.

(1) Lysis of beta cells and Collection of fractions

Collecting cultured NIT-1 cells, centrifuging, removing culture medium, resuspending NIT-1 cells with ultrapure water, freezing at-20 deg.C to-273 deg.C, adding a certain amount of ultrapure water, freeze thawing for 8 times, and subjecting to ultrasonic treatment to destroy lysed cells. After the cells are lysed, centrifuging the lysate for 15 minutes at the rotating speed of 2000g, and taking supernatant fluid, namely a water-soluble component of NIT-1 cells which can be dissolved in pure water; the precipitation fraction was dissolved by adding an aqueous solution of 8M urea (containing 500mM sodium chloride) to the obtained precipitation fraction to convert the water-insoluble fraction insoluble in pure water in the NIT-1 cells to a fraction soluble in the aqueous solution of 8M urea. And mixing the water-soluble component and the water-insoluble component according to the mass ratio of 1:1 to obtain the antigen source for preparing the control nano particles.

The NIT-1 cells were incubated for 12 hours in high-glucose medium with a doubling of glucose content and a simultaneous addition of 0.05. mu.M/L Thapsigargin to the medium. Increasing the amount of glucose or adding thapsigargin increased the amount of antigen in the beta cells. Thereafter, cultured NIT-1 cells were collected, centrifuged to remove the medium, resuspended NIT-1 cells with ultrapure water, and then frozen and thawed repeatedly 8 times with a certain amount of ultrapure water, accompanied by sonication to destroy lysed cells. After the cells are lysed, centrifuging the lysate for 15 minutes at the rotating speed of 2000g, and taking supernatant fluid, namely a water-soluble component of NIT-1 cells which can be dissolved in pure water; the precipitation fraction was dissolved by adding an aqueous solution of 8M urea (containing 500mM sodium chloride) to the obtained precipitation fraction to convert the water-insoluble fraction insoluble in pure water in the NIT-1 cells to a fraction soluble in the aqueous solution of 8M urea. Mixing the water-soluble component and the water-insoluble component according to the mass ratio of 1:1 to obtain the antigen source for preparing the nano particles for activating the antigen presenting cells.

(2) Preparation of nanoparticles

In this example, the nanoparticles and the control nanoparticles were prepared by a multiple emulsion method. The molecular weight of the adopted nano particle preparation material PLGA is 24KDa-38KDa, and the adopted inhibitors are rapamycin and everolimus. After loading the lysis fraction and immunosuppressants internally, 100mg of nanoparticles were centrifuged at 12000g for 20 minutes, resuspended in 10mL of ultrapure water containing 4% trehalose, and lyophilized for 48h for use. The average particle diameter of the nano particles is about 260nm, and the surface potential of the nano particles is about-5 mV; each 1mg of PLGA nanoparticle is loaded with about 80 mug of protein and polypeptide components, and each 1mg of PLGA nanoparticle is loaded with 0.02mg of rapamycin and everolimus respectively.

(3) Preparation of DCs

The DC is a mixed DC of peripheral blood-derived DC and BMDC. The preparation method of the two is the same as above.

(4) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (800. mu.g) were incubated with peripheral blood-derived DCs (800 ten thousand) and BMDCs (800 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO ₂ ) The incubation system contained IL-13(500U/mL), IL-10(2000U/mL), IL-4(1000U/mL) and TGF-. beta.s (2000U/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The incubated peripheral blood-derived DCs (800 ten thousand) and BMDCs (800 ten thousand) were collected by centrifugation at 400g for 5 minutes, then the cells were washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing a protease inhibitor, and after resuspension in PBS water, treated at 1000rpm for 3 minutes using a homogenizer after 1 minute of low power (20W) sonication at 4 ℃. Then centrifuging the sample at 3000g for 15 minutes and collecting supernatant, centrifuging the supernatant at 8000g for 15 minutes and collecting supernatant, filtering and extruding the supernatant by using a filter membrane of 0.22 mu m and collecting filtrate, filtering and extruding the filtrate and corresponding nano particles (30mg) prepared in the step 2 in the example 1 and DSPE-PEG-CD32 monoclonal antibody (30 mu g) at 1200RPM for 2 minutes, filtering and extruding by using a filter membrane of 0.45 mu m and collecting filtrate, centrifuging the filtrate at 15000g for 30 minutes, collecting and discarding supernatant and precipitate, and re-suspending the precipitate in PBS to obtain the nano vaccine with the particle size of 280 nanometers.

(6) Nano vaccine for preventing type I diabetes

The same as in example 1.

(7) Results of the experiment

As shown in fig. 11, the rate of type I diabetes in mice treated with the nano-vaccine prepared from the nanoparticle-activated antigen-presenting cells loaded with whole-cell antigen was significantly reduced compared to the mice in the PBS control group. Moreover, the effect of the nano-vaccine prepared from the nano-particle activated antigen presenting cells prepared from the beta cells treated by high sugar and chemical substances is better than that of the nano-vaccine prepared from the nano-particle activated antigen presenting cells prepared from the treated beta cells. Therefore, the nano vaccine has a prevention effect on type I diabetes, and the proper treatment of beta cells to increase the antigen content is favorable for improving the curative effect of the nano vaccine prepared from activated antigen presenting cells.

EXAMPLE 11 Nanoprotein made by activated antigen presenting cells for the prevention of type I diabetes

(1) Lysis of beta cells and Collection of fractions

Collecting cultured NIT-1 cells, centrifuging, removing culture medium, resuspending NIT-1 cells with ultrapure water, freezing at-20 deg.C to-273 deg.C, adding a certain amount of ultrapure water, freeze thawing for 8 times, and subjecting to ultrasonic treatment to destroy lysed cells. After cell lysis, adding nuclease (2mg/mL), acting at 37 ℃ for 30 minutes, then acting at 95 ℃ for 5 minutes to inactivate the nuclease, centrifuging the lysate for more than 1 minute at a rotating speed of more than 100g, and taking supernatant, namely a water-soluble component of NIT-1 cells which can be dissolved in pure water; the precipitation fraction was dissolved by adding an aqueous solution of 8M urea (containing 500mM sodium chloride) to the obtained precipitation fraction to convert the water-insoluble fraction insoluble in pure water in the NIT-1 cells to a fraction soluble in the aqueous solution of 8M urea. And mixing the water-soluble component and the water-insoluble component according to the mass ratio of 1:1 to obtain the antigen source for preparing the control nano particles.

(2) Preparation of nanoparticles

In this example, the nanoparticles and the control nanoparticles were prepared by multiple emulsion method. The molecular weight of PLGA used as a nano particle preparation material is 24-38 KDa, and rapamycin and tacrolimus are used as inhibitors. After loading lysis fractions and immunosuppressants internally, 100mg of nanoparticles were centrifuged at 12000g for 20min, resuspended in 10mL of ultrapure water containing 4% trehalose, and lyophilized for 48h for use. The average particle diameter of the nano particles is about 250nm, and the surface potential of the nano particles is about minus 5 mV; each 1mg of PLGA nanoparticle is loaded with about 80 mug of protein and polypeptide components, and each 1mg of PLGA nanoparticle is loaded with 0.02mg of rapamycin and tacrolimus respectively.

(3) Preparation of DC

The DC is a mixed DC of a DC derived from peripheral blood and a BMDC. The preparation method of the two is the same as above.

(4) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (800. mu.g) were incubated with peripheral blood-derived DCs (1000 million) and BMDCs (1000 million) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO2) in an incubation system containing IL-13(500U/mL), IL-10(2000U/mL), IL-4(1000U/mL) and TGF-. beta.s (2000U/mL). Alternatively, whole-cell antigen-loaded nanoparticles (800 μ g) were incubated with peripheral blood-derived DCs (800 ten thousand) and BMDCs (800 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO2), but no cytokine was added to the incubation system.

(5) Preparation of antigen presenting cell derived nano vaccine

Incubated BMDCs or BMDM were collected by centrifugation at 400g for 5 minutes, washed three times by centrifugation at 1200rpm for 3min in 30mM pH 7.0Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol, and the antigen presenting cells were then ultrasonically mechanically disrupted in the presence of phosphatase and protease inhibitors. After centrifugation, the resulting cell membranes were washed with a solution of 10mM Tris-HCl, pH 7.5, and 1mM EDTA. Then filtering the sample by membranes with the aperture of 30 μm,10 μm,5 μm, 2 μm and 0.45 μm in sequence, centrifuging the filtrate for 25 minutes at 12000g, removing supernatant, collecting precipitate, resuspending the precipitate in physiological saline containing 4% mannitol, and freeze-drying to obtain the nano vaccine with the particle size of 260 nm.

(6) Nano vaccine for preventing type I diabetes

The same as in example 1.

(7) Results of the experiment

As shown in fig. 12, the proportion of type I diabetes in the mice treated with the nano-vaccine prepared from the nanoparticle-activated antigen-presenting cells loaded with whole-cell antigen was significantly reduced compared to the mice of the PBS control group. Moreover, the effect of the nano vaccine prepared by adding the cytokine or antibody activated antigen presenting cells in the process of the nanoparticle activated antigen presenting cells is better than that of the nano vaccine prepared by adding no cytokine or antibody in the process of the nanoparticle activated antigen presenting cells. Therefore, the nano vaccine has a prevention effect on type I diabetes, and in addition, the addition of the cytokine and/or the antibody in the activation process of the antigen presenting cells is beneficial to improving the curative effect of the nano vaccine prepared by the activated antigen presenting cells.

EXAMPLE 12 Nanoprotein preparation of activated antigen presenting cells for the prevention of type I diabetes

(1) Lysis of beta cells and Collection of fractions

Collecting cultured NIT-1 cells, centrifuging, removing culture medium, resuspending NIT-1 cells with ultrapure water, freezing at-20 deg.C to-273 deg.C, adding a certain amount of ultrapure water, freeze thawing for 8 times, and subjecting to ultrasonic treatment to destroy lysed cells. After cell lysis, adding nuclease (2mg/mL), acting at 37 ℃ for 30 minutes, then acting at 95 ℃ for 5 minutes to inactivate the nuclease, centrifuging the lysate for more than 1 minute at a rotating speed of more than 100g, and taking supernatant, namely a water-soluble component of NIT-1 cells which can be dissolved in pure water; the insoluble fraction insoluble in pure water in NIT-1 cells was converted to soluble in an 8M aqueous urea solution by adding an 8M aqueous urea solution (containing 500mM sodium chloride) to the resulting precipitate fraction to dissolve the precipitate fraction. And mixing the water-soluble component and the water-insoluble component according to the mass ratio of 1:1 to obtain the antigen source for preparing the control nano particles.

(2) Preparation of nanoparticles

In this example, the nanoparticles and the control nanoparticles were prepared by a multiple emulsion method. The molecular weight of PLGA used as a nano particle preparation material is 24KDa-38KDa, the adopted inhibitors are rapamycin and interleukin 2, and KALA polypeptide (WEAKLAKALAKALAKHLAKALAKALKACEA) is used as lysosome escape substance. After loading lysis components, lysosome escaping material and immunosuppressant inside, 100mg of nanoparticles were centrifuged at 12000g for 20 minutes, resuspended with 10mL of ultrapure water containing 4% trehalose, and then lyophilized for 48 hours for use. The average particle diameter of the nano particles is about 250nm, and the surface potential of the nano particles is about-5 mV; about 80 mug of protein and polypeptide components are loaded on each 1mg of PLGA nano particles, each 1mg of PLGA nano particles are respectively loaded with 0.02mg of rapamycin and interleukin 2, and each 1mg of PLGA nano particles are respectively loaded with 0.03mg of KALA polypeptide. The preparation material and the preparation method of the contrast nano particle are the same, the average particle size is about 250nm, and the surface potential is about minus 5 mV; about 80 mug of protein and polypeptide components are loaded in each 1mg of PLGA nano particles, and each 1mg of PLGA nano particles are loaded with 0.03mg of rapamycin and interleukin 2 respectively, and are not loaded with any substance for increasing lysosome escape.

(3) Preparation of DCs

(4) Activation of antigen presenting cells

Whole cell antigen-loaded nanoparticles (800. mu.g) were incubated with peripheral blood-derived DCs (800 million) and BMDCs (800 million) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO2) in an incubation system containing IL-13(500U/mL), IL-10(2000U/mL), IL-4(1000U/mL) and TGF-. beta.s (2000U/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

Incubated BMDCs or BMDM were collected by centrifugation at 400g for 5 minutes, washed three times by centrifugation at 1200rpm for 3min in 30mM pH 7.0Tris-HCl buffer containing 0.0759M sucrose and 0.225M mannitol, and the antigen presenting cells were then ultrasonically mechanically disrupted in the presence of phosphatase and protease inhibitors. After centrifugation, the resulting cell membranes were washed with a solution of 10mM Tris-HCl, pH 7.5, and 1mM EDTA. And then sequentially filtering the sample through membranes with the aperture of 30 microns, 10 microns, 5 microns, 2 microns and 0.45 microns, centrifuging the filtrate for 25 minutes at 12000g, removing supernatant, collecting precipitate, resuspending the precipitate in physiological saline containing 4% mannitol, and freeze-drying to obtain the nano vaccine with the particle size of 260 nanometers.

(6) Nano vaccine for preventing type I diabetes

The same as in example 1.

(7) Results of the experiment

As shown in fig. 13, the proportion of type I diabetes in the nano-vaccine treated mice prepared from whole-cell antigen loaded nanoparticle-activated antigen-presenting cells was significantly reduced compared to PBS control mice. Moreover, the effect of the nano vaccine prepared by the antigen presenting cells activated by the nanoparticles loaded with the KALA polypeptides is better than that of the nano vaccine prepared by the antigen presenting cells activated by the nanoparticles not loaded with the KALA polypeptides. Therefore, the nano vaccine has a prevention effect on type I diabetes, and the KALA polypeptide is added into the nano particles or the micro particles for activating the antigen presenting cells, so that the curative effect of the nano vaccine prepared by the activated antigen presenting cells is improved. The KALA polypeptide is added in the embodiment, and one or more of other substances with lysosome escape increasing function can be loaded in the actual use process, such as arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH ₄ HCO ₃ Polypeptides with positive charges, protamine, histone and the like.

Example 13 Nanoprotein vaccine prevention of autoimmune diseases

(1) Lysis of beta cells

The glucose content was doubled in high-sugar medium and NIT-1 cells were incubated for 12 hours with 0.05. mu.M/L Thapsigargin added to the medium. Increasing the amount of glucose and adding thapsigargin increased the amount of antigen in the beta cells. Then, collecting the cultured NIT-1 cells, removing the culture medium after centrifugation, re-suspending and lysing the NIT-1 cells by using 10% sodium deoxycholate aqueous solution (containing 8M arginine), and dissolving the lysate component by using 10% sodium deoxycholate aqueous solution (containing 8M arginine) to obtain the antigen source of the nano particles for preparing the activated antigen presenting cells.

(2) Preparation of nanoparticles

In the embodiment, the nanoparticles are prepared by a solvent volatilization method, the molecular weight of PLGA (polylactic-co-glycolic acid) serving as a nanoparticle preparation material is 38-54 KDa, the adopted immunosuppressants are rapamycin and mycophenolate mofetil, the adopted substances for increasing the escape of lysosomes are polyarginine and RALA polypeptides, and the immunosuppressants, the polyarginine and the RALA polypeptides are all loaded in the nanoparticles. The preparation method is as described above, in the preparation process, firstly, the components, the immunosuppressant, the polyarginine and the RALA polypeptide are cracked in the nanoparticles by a multiple emulsion method, then 100mg of PLGA nanoparticles are centrifuged at 13000g for 20min, the precipitate is resuspended by 4% trehalose, and then the precipitate is frozen and dried for 48 hours for later use. The average particle diameter of the nano particles is about 260 nm; each 1mg PLGA nanoparticle was loaded with about 140. mu.g of protein or polypeptide component, 0.03mg each of rapamycin and mycophenolate mofetil, and 0.02mg each of polyarginine and RALA polypeptides.

The blank nano-particles are prepared by the same material and preparation method, the average particle diameter of the blank nano-particles is about 260nm, each 1mg PLGA blank nano-particles are loaded with 0.03mg of rapamycin and mycophenolate mofetil respectively, and are loaded with 0.02mg of polyarginine and RALA polypeptide respectively, but no lysate component is loaded.

(3) Activation of antigen presenting cells

In this example, DC2.4 cells, B cells derived from splenocytes, and BMDM were used as mixed antigen-presenting cells, and three antigen-presenting cells were prepared as described above. DC2.4 cells, B cells and BMDM were mixed in a quantitative ratio of 1:1: 1.1 mg of whole-cell antigen-loaded nanoparticles were incubated with 3000 ten thousand mixed antigen-presenting cells (1000 ten thousand DCs +1000 ten thousand B cells +1000 ten thousand BMDM) in 25mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO ₂ ) The incubation system contained IL-7(100U/mL), IL-10(1000U/mL), IL-4(500U/mL) and TGF-. beta.s (1000U/mL). After the incubation is finished, the cell sediment is collected by centrifugation at 400g for 5 minutes, and the mixed antigen presenting cells are collected for standby after two times of centrifugation and washing by PBS. The activated mixed antigen presenting cell cells are used as a live cell vaccine 3.

(4) Preparation of nano-vaccine

The glucose content was doubled in high-sugar medium and NIT-1 cells were incubated for 18 hours with 0.05. mu.M/L Thapsigargin added to the medium. Thereafter, the cultured NIT-1 cells were collected. 3000 ten thousand NIT-1 cells were mixed with 3000 ten thousand step (3) activated mixed antigen presenting cells (1000 ten thousand DCs +1000 ten thousand B cells +1000 ten thousand BMDM), and then the mixed cells were washed twice with 4 ℃ Phosphate Buffered Saline (PBS) containing a protease inhibitor, and the cells were resuspended in PBS water followed by low power (10W) sonication at 4 ℃ for 20 minutes. Then, the sample is centrifuged at 3000g for 15 minutes and the supernatant is collected, the supernatant is filtered through membranes with the pore diameters of 30 μm,10 μm,5 μm, 2 μm,1 μm,0.45 μm and 0.22 μm in sequence, the filtrate is collected, then the filtrate is centrifuged at 18000g for 50 minutes, the supernatant is discarded and the precipitate is collected, and the precipitate is resuspended in PBS to obtain a mixed cell membrane component. And (3) stirring the mixed cell membrane component and the nanoparticles (100mg) loaded with the whole cell component prepared in the step (2) or blank nanoparticles (100mg) at 1500RPM for 5 minutes, then co-incubating for 15 minutes at room temperature, filtering and extruding by using a filter membrane of 0.45 mu m, then centrifuging for 25 minutes at 13000g, then re-suspending by using 10mL of a freeze-drying protective agent (containing 2% trehalose, 2% mannitol and 1% sucrose) aqueous solution, and then freezing and drying for 48 hours to obtain the nano vaccine. Wherein, the nano vaccine obtained by the combined action of the nano particles loaded with the whole cell components and the mixed cell membrane components is nano vaccine 1, and the particle size of the nano vaccine 1 is 270 nanometers; the nano vaccine obtained by the combined action of the blank nano particles and the mixed cell membrane components is nano vaccine 2, and the particle size is 270 nanometers.

(5) Nano vaccine for preventing type I diabetes

The control group of the study was the PBS group. Female NOD mice of 3 weeks of age were selected for this experiment. In the experiment, 10 NOD mice per group were injected subcutaneously with 100 μ g of nano-

vaccine

1, or 100 μ g of nano-vaccine 2, or 300 ten thousand mixed antigen presenting cell vaccine 3(100 ten thousand DCs +100 ten thousand B cells +100 ten thousand BMDMs), or 100 μ L of PBS for 6 consecutive weeks starting from the third week every 7 days. Mice blood glucose was recorded daily for each group of mice starting at week 8. Onset of diabetes is determined by blood glucose higher than 11.0 mmol.L-1. And recording the diabetes onset of NOD mice at different time periods.

(6) Antigen-specific regulatory T cells (T) _reg ) Analysis of (2)

Female C57BL/6 at 6-8 weeks was selected as model mice, and 100. mu.g of Nanoprotein 1, or Nanoprotein 2, or 300 million mixed antigen presenting cell vaccines 3(100 million DCs +100 million B cells +100 million BMDMs), or PBS was injected subcutaneously into each mouse on days 0, 7, 14, 28, and 42, respectively. On day 45, the mice were sacrificed, spleens were removed and single cell suspensions of splenocytes were prepared, and B cells and T cells were sorted from the mouse splenocytes using magnetic bead sorting. Mu.g of nanoparticles loaded with beta cell whole cell antigen, 500 ten thousand B cells and 100 ten thousand T cells were CO-incubated in 5mL of RPMI1640 complete medium for 48 hours (37 ℃, 5% CO) ₂ ). The incubated cells were then collected and labeled with live and dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody and FOXP3 antibody, and the T cell subpopulations were then analyzed by flow cytometry for CD4 ⁺ CD25 ⁺ FOXP3 ⁺ T cell occupancy CD4 ⁺ Ratio of T cells and CD8+ Ly49 ⁺ T accounts for CD8 ⁺ Proportion of T cells.

(7) Results of the experiment

As shown in fig. 14a, the vaccine treated mice all had a significantly reduced rate of type I diabetes compared to the PBS group. Moreover, the effect of the nano vaccine 1 is obviously better than that of the nano vaccine 2 and the live cell vaccine 3. The results show that the nano vaccine internally loaded with the whole cell antigen and simultaneously surface-loaded with the mixed cell membrane component containing the antigen and the activated antigen presenting cell is better than the nano vaccine internally not loaded with the antigen and only surface-loaded with the mixed cell membrane component, and also that the nano vaccine internally loaded with the immunosuppressant and the lysosome escape substance and simultaneously surface-loaded with the mixed cell membrane component containing the antigen and the activated antigen presenting cell is better than the mixed antigen presenting cell live cell vaccine activated by the nano particles loaded with the whole cell antigen. In conclusion, the nano vaccine loaded with the mixed cell membrane component on the surface and internally loaded with the whole cell antigen has a good prevention effect on the type I diabetes.

As shown in FIGS. 14b and 14c, the nano-vaccine 1 can induce autoantigen-specific T _reg More than nano vaccine 2 and nano vaccine 3. This shows that the internal loading of cell whole cell antigen and the surface loading of cell membrane containing mixed cells are beneficial to inducing more autoantigen specific T _reg . Because T having cell killing ability can be inhibited _eff Therefore, the induced can be effective in preventing or treating type I diabetes.

In this embodiment, the membrane component of the cell membrane of the cell containing the autoantigen is loaded on the surface of the nano vaccine, and in practical application, the membrane component of the extracellular vesicle secreted by the cell containing the autoantigen can also be used.

EXAMPLE 14 Nanoprotein for treatment of type I diabetes

(1) Lysis of beta cells

Cultured NIT-1 cells were centrifuged at 400g for 5 minutes, then washed twice with PBS and resuspended in ultrapure water. And (3) inactivating and denaturing the obtained cells by respectively adopting ultraviolet rays and high-temperature heating, then adding ultrapure water, repeatedly freezing and thawing for 5 times, assisting ultrasonic cell lysis, then adding nuclease for acting for 5 minutes, and acting for 10 minutes at 95 ℃ to inactivate the nuclease. Centrifuging the cell lysate for 10 minutes at 5000g, wherein the supernatant is a water-soluble component, dissolving the precipitate with 10% octyl glucoside to obtain a dissolved original water-insoluble antigen, and mixing the water-soluble component and the water-insoluble component according to the mass ratio of 2:1 to obtain the lysate component required for preparing the micron particles.

(2) Preparation of microparticle systems

In this example, the microparticle system was prepared and the control microparticle was prepared by multiple emulsion method, the molecular weight of PLGA, the microparticle matrix material, was 38kDa-54kDa, the immunosuppressant used was methylprednisolone, and the lysosomal escape-increasing substance used was NH ₄ HCO ₃ And melittin. During preparation, firstly, the internal load lysate component, methylprednisolone and NH are prepared by a multiple emulsion method ₄ HCO ₃ And melittin microparticles, then 100mg microparticles were centrifuged at 9000g for 20 minutesAfter 10mL of ultrapure water containing 4% trehalose was resuspended, the suspension was dried for 48 hours for use. The average particle diameter of the micron particles is about 3.1 mu m, and the surface potential is about-7 mV; each 1mg PLGA microparticle was loaded with about 110. mu.g of protein or polypeptide fraction, containing methylprednisolone 0.01mg, NH4HCO3 and melittin 0.02mg each. The preparation material and the preparation method of the reference micrometer particles are the same as the above method, the average particle size of the reference micrometer particles is about 3.1 μm, and the surface potential is about-7 mV; every 1mg PLGA microparticle is loaded with about 110 μ g protein or polypeptide component, no immunosuppressant, and only NH ₄ HCO ₃ And melittin 0.02mg each.

(3) Preparation of antigen-presenting cells

This example uses the DC2.4 cell line as an antigen presenting cell.

(4) Activation of antigen presenting cells

Cancer cell Whole cell antigen-loaded microparticles (1000. mu.g) were incubated with DC2.4(1000 ten thousand) in 15mL of high-glucose DMEM complete medium for 48 hours (37 ℃, 5% CO) ₂ ) (ii) a The incubation system contained GM-CSF (2000U/mL), IL-2(100U/mL), IL-10(2000U/mL), and IL-13 (200U/mL).

(5) Preparation of antigen presenting cell derived nano vaccine

The post-incubation DCs were collected by centrifugation at 400g for 5 minutes, followed by washing the cells twice with 4 ℃ Phosphate Buffered Saline (PBS) containing protease inhibitors, resuspending the cells in PBS water followed by 2 minutes of low power (20W) sonication at 4 ℃. Then centrifuging the sample at 3000g for 15 minutes, collecting the supernatant, filtering the supernatant sequentially through membranes of 30 μm,10 μm,5 μm,1 μm and 0.45 μm, and centrifuging at 15000g for 60 minutes to obtain the nano vaccine, wherein the average particle size of the nano vaccine is 250 nanometers.

(5) Nano vaccine for treating type I diabetes

The same as in example 7.

(6) Results of the experiment

As shown in fig. 15, the nano-vaccine treated type I mice prepared from the microparticle-activated antigen-presenting cells were improved after treatment compared to the PBS control group. Moreover, the nano-vaccine prepared by the micron particle activated antigen presenting cells loaded with the immunosuppressant and the lysosome escaping substance simultaneously is superior to the nano-vaccine prepared by the micron particle activated antigen presenting cells loaded with the lysosome escaping substance only and not loaded with the immunosuppressant. The immune inhibitor can improve the efficiency of the micron particles for activating the antigen presenting cells, and is beneficial to better induce the T cells to be converted into the regulatory T cells with specific immune inhibition function by the nano vaccine prepared from the antigen presenting cells.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A method of preparing an autoimmune disease vaccine derived from a pre-activated antigen presenting cell, comprising the steps of:

wherein the content of the first and second substances,

the first delivery particle or the second delivery particle is independently a nanoparticle or a microparticle, respectively;

the autoimmune disease whole cell antigen is prepared by the following steps: freezing cells or tissues containing the autoimmune disease antigen, adding water for freeze-thaw lysis, collecting supernatant and precipitate, dissolving by a dissolving agent, and converting into a soluble part to obtain the autoimmune disease whole cell antigen; or adding a lytic agent into cells or tissues containing the autoimmune disease antigen for cracking, and collecting soluble parts to obtain the autoimmune disease whole cell antigen.

2. The method of claim 1, wherein: loading the cell membrane fraction of the pre-activated antigen-presenting cells onto a second delivery particle loaded with an autoimmune disease whole cell antigen, and simultaneously loading the cell membrane fraction of cells containing autoimmune disease antigen onto the second delivery particle.

3. The method of claim 1, wherein: in step S1, the incubation system contains cell factors and/or antibodies during co-incubation; the cytokine is selected from one or more of interleukin 1 receptor antagonist, growth factor, interleukin, interferon, tumor necrosis factor, colony stimulating factor, activin and inhibin; the antibody is selected from one or more of a PD1 antibody, a PD-L1 antibody, a CTAL-4 antibody, a TIGIT antibody, a TIM-3 antibody, a LAG-3 antibody, an alpha CD-8 antibody, an alpha CD-28 antibody, an alpha CD-40 antibody, an alpha OX-40 antibody, and an alpha OX-40L antibody.

4. The method of claim 1, wherein: the dissolving agent is selected from one or more of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salt, Triton, Tween, amino acid, glucoside and choline.

5. The method of claim 1, wherein: in step S1, the incubation system contains glucose and/or thapsigargin during co-incubation.

6. The method of claim 1, wherein: the antigen presenting cell is selected from at least one of a dendritic cell, a B cell, and a macrophage.

7. The method of claim 1, wherein: the first delivery particle or the second delivery particle is loaded with an immunity-inhibiting substance; the substance for inhibiting the immunity is selected from one or more of mRNA, DNA, glucocorticoid drugs, calcineurin inhibitors, antimetabolites, antibodies, cytokines, alkylating agents, cyclosporine, rapamycin, tacrolimus, guanrolimus, fingolimod, methylprednisolone, tripterygium wilfordii, mycophenolate mofetil, cyclophosphamide, azathioprine, everolimus, climax, setipine, cyclosporine A, novamin, TGF-beta, interleukins, ginseng and astragalus membranaceus.

8. The method of claim 1, wherein: the first delivery particle or the second delivery particle is loaded with KALA polypeptide, RALA polypeptide, melittin, arginine, polyarginine, lysine, polylysine, histidine, polyhistidine, NH ₄ HCO ₃ One or more of protamine and histone.

9. An autoimmune disease vaccine prepared by the method of any one of claims 1 to 8.

10. Use of the autoimmune disease vaccine of claim 9 in the manufacture of a medicament for the treatment or prevention of an autoimmune disease.