WO2023236331A1 - 一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法及其应用 - Google Patents

一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法及其应用 Download PDF

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WO2023236331A1
WO2023236331A1 PCT/CN2022/108962 CN2022108962W WO2023236331A1 WO 2023236331 A1 WO2023236331 A1 WO 2023236331A1 CN 2022108962 W CN2022108962 W CN 2022108962W WO 2023236331 A1 WO2023236331 A1 WO 2023236331A1
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cells
antigen
cell
nanoparticles
loaded
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刘密
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苏州尔生生物医药有限公司
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
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    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of immunotherapy, and in particular to a preparation method and application of an autoimmune disease vaccine derived from preactivated antigen-presenting cells.
  • Autoimmune diseases are a type of disease that seriously threaten human health.
  • the pathogenesis is that the human immune system mistakes its own substances for foreign antigens for some reasons and launches attacks on cells or tissues containing such antigens, leading to a series of consequences and disease.
  • type I diabetes as an example, for some reasons that are not fully understood, the body's immune system mistakes certain components in the pancreatic islets or beta cells as foreign antigens, thereby launching an attack on the beta cells, and then killing most of the beta cells. cell. Since beta cells are the substance that secretes insulin in the human body, and insulin is the key substance to control blood sugar, when most of the beta cells are killed, the body cannot produce enough insulin to control blood sugar, and type I diabetes occurs.
  • the invention provides a method for preparing an autoimmune disease vaccine derived from preactivated antigen-presenting cells, which includes the following steps:
  • the first delivery particles or the second delivery particles are independently nanoparticles or microparticles
  • the autoimmune disease whole cell antigen is prepared by the following steps: freezing the cells or tissues containing the autoimmune disease antigen, adding water for freeze-thaw lysis, collecting the supernatant and the soluble part of the precipitate after being dissolved by a dissolving agent , obtain the autoimmune disease whole cell antigen; or add a dissolving agent to cells or tissues containing the autoimmune disease antigen for lysis, and collect the soluble parts to obtain the autoimmune disease whole cell antigen.
  • the dissolving agent is selected from the group consisting of urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, inorganic salts, Triton, Tween, amino acids, glycosides and choline one or more of them.
  • delivery particles all refer to the first delivery particles or the second delivery particles
  • delivery particles simultaneously load the membrane component of the antigen-presenting cells and the membrane component of the cells containing the autoimmune disease antigen (hereinafter referred to as
  • the specific steps include:
  • Cells containing autoimmune disease antigens are subjected to mechanical destruction, membrane filtration, gradient centrifugation or chemical treatment to obtain cell membrane fragments and/or nanovesicles derived from ⁇ cells;
  • the mechanical destruction method is selected from one or more of ultrasonic, homogenization, homogenization, high-speed stirring, high-pressure destruction, high-shear force destruction, swelling, and shrinkage.
  • the co-action mode is selected from one or more of co-incubation, ultrasound, co-extrusion, ultrafiltration, dialysis, stirring, homogenization and homogenization.
  • cytokines include but are not limited to interleukin 1 receptor antagonist (IL-1ra), interleukin 2 (IL-2), transforming growth factor- ⁇ (TGF- ⁇ ), 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- ⁇ ), TNF- ⁇ , granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), activins, and inhibins.
  • IL-1ra interleukin 1 receptor antagonist
  • IL-2 interleukin 2
  • TGF- ⁇ transforming growth factor- ⁇
  • IL-7 inter
  • the incubation system includes one of the following combinations:
  • GM-CSF IL-2, IL-7, IL-10 and CD40 antibodies
  • TGF- ⁇ , IL-4, IL-7, IL-10 (most preferred);
  • TGF- ⁇ , IL-10, IL-4 and PD-L1 antibodies (4) TGF- ⁇ , IL-10, IL-4 and PD-L1 antibodies;
  • step S1 during co-incubation, the incubation system contains glucose and/or thapsigargin.
  • the antigen presenting cell is selected from at least one of dendritic cells (DC), B cells and macrophages.
  • the nano vaccine or micro vaccine of the present invention is characterized in that: when the autoimmune disease is type I diabetes, the cells containing antigens related to type I diabetes are ⁇ cells; the tissue is a tissue containing ⁇ cells, such as Pancreatic tissue and/or islet tissue.
  • the activated antigen-presenting cells can be washed appropriately before preparing the nanovaccine, and the washing solution used in the washing process contains protease inhibitors.
  • the process of ultrasonic treatment of antigen-presenting cells for preparing nano-vaccines or micro-vaccines is low-power ultrasound (less than 500W);
  • the gradient centrifugation for preparing nano-vaccines or micro-vaccines is gradient centrifugation with centrifugal speeds increasing in sequence;
  • the pore sizes of the filter membranes used for membrane filtration of micron vaccines are in order from large to small; the total action time is greater than 20 seconds.
  • the particle size of nano vaccine is greater than 30 nm and less than 1000 nm; the particle size of micron vaccine is greater than 1 ⁇ m and less than 50 ⁇ m.
  • the particle size of nanoparticles used to activate antigen-presenting cells ranges from 10 nm to 1000 nm, and the particle size of microparticles used to activate antigen-presenting cells ranges from 1 ⁇ m to 50 ⁇ m.
  • the interior and/or surface of the first delivery particle or the second delivery particle is loaded with an immune-suppressing substance.
  • first delivery particle or the second delivery particle is loaded with positively charged polypeptides (such as KALA polypeptide, RALA polypeptide, melittin, etc.), arginine, polyarginine, lysine, polylysine One or more of acid, histidine, polyhistidine, NH 4 HCO 3 , protamine and histone.
  • positively charged polypeptides such as KALA polypeptide, RALA polypeptide, melittin, etc.
  • the present invention also provides an application of the above-mentioned autoimmune disease vaccine in the preparation of drugs for treating or preventing autoimmune diseases.
  • nanoparticles or microparticles are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials, and can be prepared using existing preparation methods, including but not limited to common solvent evaporation methods, dialysis methods, and microfluidics. Control method, extrusion method, hot melt method.
  • organic synthetic polymer materials include but are not limited to polylactic acid-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, polymethyl methacrylate (PMMA) ), polyamino acids, synthetic peptides, etc.; natural polymer materials include lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane components, starch, sugars, peptides, etc.; inorganic materials include iron oxide, Ferric oxide, carbonates, phosphates, etc.
  • the size of the particles used to activate the antigen-presenting cells is nanometer or micron, which can ensure that the particles are phagocytized by the antigen-presenting cells.
  • the particle size In order to improve the phagocytosis efficiency, the particle size must be within an appropriate range.
  • the particle size of nanoparticles is 1nm-1000nm, more preferably, the particle size is 30nm-1000nm, most preferably, the particle size is 50nm-600nm; the particle size of microparticles is 1 ⁇ m-1000 ⁇ m, more preferably,
  • the particle size is 1 ⁇ m-100 ⁇ m, more preferably, the particle size is 1 ⁇ m-10 ⁇ m, and most preferably, the particle size is 1 ⁇ m-5 ⁇ m.
  • the vaccine can integrate one or more components of antigen-presenting cells and antigenic epitopes of autoimmune diseases into a nano-vaccine or micro-vaccine.
  • a nano-vaccine or micro-vaccine can contain DC cells, A component of at least one antigen-presenting cell such as B cells or macrophages, so a nano-vaccine or micro-vaccine can have certain functions and advantages of one or more antigen-presenting cells at the same time.
  • Nano-vaccines or micro-vaccines can contain components of one or more antigen-presenting cells including DC cells. After injection into the body, they have the characteristics of homing to lymph nodes and can better activate antigen-specific inhibitory properties. immune response.
  • nanoparticles and/or microparticles loaded with whole-cell antigens of cells and/or tissues containing autoimmune disease antigens to activate antigen-presenting cells, and then use the antigen-presenting cells to prepare nanovaccines. Therefore, the prepared nanoparticles Vaccines can be loaded with all antigenic epitopes loaded by nanoparticles and/or microparticles used to activate antigen-presenting cells, because nanovaccines can activate a broad spectrum of regulatory antigen-specific T cells (Treg).
  • Treg regulatory antigen-specific T cells
  • Nano-vaccines or micro-vaccines are derived from antigen-presenting cells, and their components are biocompatible and degradable, with good safety.
  • nano-vaccines or micro-vaccines are not as easy to store, transport and inject as live antigen-presenting cells. The requirements are so stringent.
  • the present invention In addition to wrapping the membrane components of antigen-presenting cells on delivery particles, the present invention also loads the membrane components of cells containing self-antigens on the surface of nanoparticles or microparticles, thus endowing nanovaccines or The ability of micron vaccines to load broad-spectrum antigens and the ability to have a cell membrane structure on the surface can achieve some effects that cannot be achieved without bionic membrane particles through the effect of surface-loaded bionic membranes.
  • Figure 1 is a schematic diagram of the preparation process and application of the nano-vaccine or micro-vaccine of the present invention
  • a is the collection of water-soluble components and non-water-soluble components in the whole cell antigen after lysing cells or tissues containing autoimmune disease antigens, and then Schematic diagram of preparing nanoparticles or microparticles
  • b is a schematic diagram of using a dissolving solution containing a dissolving agent to lyse and dissolve whole cell antigens of cells or tissues containing autoimmune disease antigens and preparing nanoparticles or microparticles
  • c is a schematic diagram of using a Or, after activating the antigen-presenting cells with the above-mentioned particles prepared in step b, the antigen-presenting cells are prepared into nano-vaccines or micro-vaccines, and the nano-vaccines are used to prevent or treat autoimmune diseases such as type I diabetes;
  • Figures 2-15 are respectively the experimental results when nano vaccines or micron vaccines are used to prevent or treat autoimmune diseases such as type I diabetes in Examples 1-14; in Figures 3, 4 and 14, a is the prevention or treatment of type I diabetes, etc.
  • the results of autoimmune diseases, b and c are flow cytometry analysis of the proportion of CD8 + and CD4 + regulatory specific T cells activated by the nanovaccine to the total CD8 + and CD4 + T cells.
  • the nano-vaccine or micro-vaccine of the present invention for preventing or treating autoimmune diseases such as type I diabetes contains nano-particles and/or micro-vaccines composed of cells loaded with autoimmune disease antigens and/or tumor tissue whole cell antigens. Nano-vaccines or micro-vaccines prepared from particle-activated antigen-presenting cells. Nanoparticles and/or microparticles are loaded with autoimmune disease antigens, cell and/or tissue whole cell antigens, or mixtures thereof. Preparation of nano-vaccines or micro-vaccines to prevent or treat autoimmune diseases such as type I diabetes. The preparation process and application fields are shown in Figure 1.
  • the cells or tissues can be lysed and then the water-soluble antigens and water-insoluble antigens can be collected separately. And prepare nanoparticle or microparticle systems respectively; or you can also directly use a dissolving solution containing a dissolving agent to directly lyse cells or tissues and dissolve whole cell antigens to prepare nanoparticle or microparticle systems.
  • the whole cell antigen of the present invention can be processed before or (and) after lysis, including but not limited to inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, nuclease treatment, etc. Then prepare nanoparticles or microparticles; you can also directly prepare nanoparticles or microparticles without any inactivation or (and) denaturation, solidification, biomineralization, ionization, chemical modification, or nuclease treatment before or after cell lysis. Micron particles.
  • tissue cells undergo inactivation or/and denaturation before lysis. In actual use, inactivation or/and denaturation can also be performed after cell lysis, or before cell lysis.
  • the inactivation or (and) denaturation treatment method before or (and) after cell lysis is ultraviolet irradiation and high-temperature heating.
  • Treatment methods including but not limited to radiation irradiation, high pressure, curing, biomineralization, ionization, chemical modification, nuclease treatment, collagenase treatment, freeze-drying and other treatment methods can also be used. Those skilled in the art can understand that during actual application, the skilled person can make appropriate adjustments according to specific circumstances.
  • cytokines and/or antibodies can be used to assist in improving the activation efficiency.
  • the antigen-presenting cells can be derived from autologous or allogeneic cells, or from cell lines or stem cells.
  • Antigen-presenting cells can be DC cells, B cells, macrophages, or any mixture of the above three, or other cells with antigen-presenting functions.
  • the antigen-presenting cells are prepared into nano- or micro-vaccines by methods such as centrifugation and/or membrane filtration and/or co-action with nanoparticles or microparticles. In actual preparation, other methods can also be used to prepare live antigen-presenting cells into nano-vaccines or micro-vaccines without cell activity.
  • the antigen-presenting cells are prepared into nano-vaccines or micro-vaccines by using nanoparticles or micron particles activated with antigen-presenting cells loaded with cell and/or tissue whole-cell antigens containing autoimmune disease antigens. Methods as below:
  • Step 1 Add a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a second predetermined concentration of the raw material for preparing particles.
  • the aqueous solution may contain each component of the cell lysate and an immunosuppressant; each component of the cell lysate is a water-soluble antigen or is dissolved in urea or guanidine hydrochloride during preparation.
  • the concentration of the water-soluble antigen or the original non-water-soluble antigen contained in the aqueous phase solution, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 1ng/mL, which can load enough whole cell antigens to activate relevant cells.
  • the concentration of immunosuppressant in the initial aqueous phase is greater than 0.01 ng/mL.
  • the aqueous solution contains each component in the tissue lysate and an immunosuppressant; each component in the tissue lysate is a water-soluble antigen or is dissolved in a dissolving agent such as urea or guanidine hydrochloride during preparation.
  • the original non-water-soluble antigen in.
  • the concentration of the water-soluble antigen or the original concentration of the non-water-soluble antigen contained in the aqueous phase solution, that is, the first predetermined concentration requires the protein polypeptide concentration to be greater than 0.01ng/mL, which can load enough whole-cell antigen to activate relevant cells.
  • the concentration of immunosuppressant in the initial aqueous phase is greater than 0.01 ng/mL.
  • the raw material for preparing particles is polylactic acid-co-glycolic acid (PLGA) or polylactic acid (PLA), and methylene chloride is used as the organic solvent.
  • the second predetermined concentration of raw materials for preparing particles ranges from 0.5 mg/mL to 5000 mg/mL, preferably 100 mg/mL.
  • the reason why PLGA, PLA or modified PLGA, PLA is selected is that the material is a biodegradable material and has been approved by the FDA for use as a pharmaceutical excipient. Studies have shown that PLGA and PLA have certain immunomodulatory functions and are therefore suitable as excipients in the preparation of nanoparticles or microparticles. In practical applications, appropriate materials can be selected according to actual conditions.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase.
  • the range of the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is It is 1:1.1-1:5000, 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 prepared nanoparticles or microparticles.
  • the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL ⁇ 100 mg/mL; when the aqueous phase solution is a lysate component/immunosuppressant solution, wherein The concentration of protein and polypeptide is greater than 1ng/mL, preferably 1mg/mL ⁇ 100mg/mL, and the concentration of immune adjuvant is greater than 0.01ng/mL, preferably 0.01mg/mL ⁇ 20mg/mL.
  • the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropyl alcohol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane;
  • the concentration of the organic phase is 0.5 mg/mL ⁇ 5000mg/mL, preferably 100mg/mL.
  • Step 2 subject the mixed solution obtained in Step 1 to ultrasonic treatment for more than 2 seconds or stirring or homogenization treatment or microfluidic treatment for more than 1 minute.
  • the stirring is mechanical stirring or magnetic stirring
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute.
  • the stirring speed is 50 rpm ⁇ 1500 rpm
  • the stirring time is 0.1 hour ⁇ 24 hours
  • the ultrasonic power is greater than 5W
  • the time Greater than 0.1 seconds such as 2 to 200 seconds
  • the pressure is greater than 5 psi, such as 20 psi to 100 psi.
  • the rotation speed of the shear homogenizer is greater than 100rpm, such as 1000rpm to 5000rpm; the flow rate of microfluidic processing is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used for nanonization and/or micronization.
  • the length of ultrasonic time or stirring speed or homogenization pressure and time can control the size of the prepared micro-nano particles. Too large or too small will cause to changes in particle size.
  • Step 3 Add the mixture obtained after step 2 to a third predetermined volume of aqueous solution containing a third predetermined concentration of emulsifier and perform ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or perform homogenization or microfluidic treatment. deal with.
  • the mixture obtained in step 2 is added to the aqueous emulsifier solution and continued to be ultrasonically or stirred to form nanometers or micrometers.
  • the ultrasonic time is greater than 0.1 seconds, such as 2 to 200 seconds
  • the stirring speed is greater than 50 rpm, such as 50 rpm to 500 rpm
  • the stirring time is greater than 1 minute, such as 60 to 6000 seconds.
  • the stirring speed is greater than 50rpm, and the stirring time is greater than 1 minute.
  • the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.5 to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W to 500W.
  • the time is greater than 0.1 seconds, such as 2 to 200 seconds; when homogenizing, use a high-pressure/ultra-high-pressure homogenizer or high-shear homogenizer.
  • the pressure is greater than 20 psi, such as 20 psi to 100 psi.
  • the rotation speed is greater than 1000rpm, such as 1000rpm ⁇ 5000rpm; when using microfluidic processing, the flow rate is greater than 0.01mL/min, such as 0.1mL/min-100mL/min.
  • Ultrasonic or stirring or homogenization treatment or microfluidic treatment can be used to nanonize or micronize the particles.
  • the length of ultrasonic time or stirring speed or homogenization process pressure and time can control the size of the prepared nano or micron particles. Too large or too small will cause Changes in particle size.
  • the emulsifier aqueous solution is poly(ethylene-alt-maleic anhydride) (PEMA) aqueous solution or polyvinyl alcohol (PVA), the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume and the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time, the volume and concentration of the emulsifier aqueous solution in this step are all based on obtaining nanoparticles or microparticles of suitable size.
  • Step 4 Add the liquid obtained after the treatment in Step 3 to a fourth predetermined volume of the emulsifier aqueous solution with a fourth predetermined concentration, and stir until the predetermined stirring conditions are met.
  • the emulsifier aqueous solution is PEMA or PVA solution or other solutions.
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles or microparticles of suitable size.
  • the selection of the fourth predetermined volume is determined based on the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume and the fourth predetermined volume can be adjusted in order to control the size of the nanoparticles or microparticles.
  • the predetermined stirring condition of this step is until the volatilization of the organic solvent is completed, that is, the volatilization of methylene chloride in step 1 is completed.
  • Step 5 After centrifuging the mixed liquid that meets the predetermined stirring conditions in Step 4 at a rotation speed of greater than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining sediment in a fifth predetermined volume of Five predetermined concentrations of an aqueous solution containing a lyoprotectant or a sixth predetermined volume of PBS (or physiological saline).
  • step 5 when the precipitate obtained in step 5 is resuspended in the sixth predetermined volume of PBS (or physiological saline), there is no need to freeze-dry, and the subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles can be directly performed.
  • PBS physiological saline
  • the precipitate obtained in step 5 needs to be freeze-dried when resuspended in an aqueous solution containing a lyoprotectant, and then freeze-dried before subsequent adsorption of cancer cell lysates on the surface of nanoparticles or microparticles. experiment.
  • Trehalose is selected as the freeze-drying protective agent.
  • Step 6 After freeze-drying the suspension containing the lyoprotectant obtained in Step 5, the freeze-dried material is used for later use.
  • Step 7 Resuspend a sixth predetermined volume of the nanoparticle-containing suspension obtained in Step 5 in PBS (or physiological saline) or use a sixth predetermined volume of PBS (or physiological saline) to resuspend the nanoparticle-containing suspension obtained in Step 6
  • PBS or physiological saline
  • the freeze-dried substance containing nanoparticles or microparticles and a lyoprotectant is used directly; or the above sample is mixed with a seventh predetermined volume of water-soluble antigen or the dissolved original non-water-soluble antigen and used.
  • Step 9 Collect the co-incubated antigen-presenting cells and/or cultured cells containing autoantigens, such as ⁇ cells, which can be washed or not washed and then treated with sonication, mechanical stirring, homogenization, etc.
  • autoantigens such as ⁇ cells
  • Step 10 The mechanically treated antigen-presenting cells and/or cell samples containing autoantigens are subjected to gradient centrifugation, and/or membrane filtration, and/or co-action with nanoparticles or microparticles to prepare nano vaccines or micron vaccines. vaccine.
  • Steps 1 to 4 are the same as above.
  • the precipitate obtained in step 6 does not need to be freeze-dried after being resuspended in the seventh predetermined volume of charged substance, and subsequent experiments related to loading cell/tissue lysate on the surface of nanoparticles or microparticles can be directly performed.
  • the freeze-drying protective agent is trehalose or a mixed solution of mannitol and sucrose.
  • concentration of the drying protective agent in this step is 4% by mass, which is set so as not to affect the drying effect during subsequent drying.
  • Step 7 After drying the suspension containing the drying protective agent obtained in Step 6, the dried material is used for later use.
  • Step 10 Collect the co-incubated antigen-presenting cells and/or cultured cells containing autoantigens, such as ⁇ cells, which can be washed or not washed and then treated with sonication, mechanical stirring, homogenization, etc.
  • autoantigens such as ⁇ cells
  • NIT-1 ⁇ cells After culturing NIT-1 ⁇ cells in high-glucose medium, collect the cultured NIT-1 ⁇ cells, remove the medium after centrifugation, resuspend the NIT-1 ⁇ cells in ultrapure water, and then freeze them at -20°C to -273°C. Add a certain amount of ultrapure water and freeze and thaw repeatedly for more than 3 times, accompanied by ultrasound to destroy the lysed cells. After the cells are lysed, centrifuge the lysate at 3000g for 5 minutes, and take the supernatant, which is the water-soluble component of NIT-1 ⁇ cells that is soluble in pure water; add 8M urea (containing 500mM sodium chloride) to the resulting precipitate.
  • 8M urea containing 500mM sodium chloride
  • This example uses the preparation of dendritic cells from mouse bone marrow cells as an example to illustrate how to prepare BMDC.
  • a 6-8 week old C57 mouse was killed by cervical dislocation.
  • the tibia and femur of the hind legs were surgically removed and placed in PBS.
  • the muscle tissue around the bones was removed with scissors and tweezers.
  • the needles are inserted into the bone marrow cavity from both ends of the bone, and the bone marrow is repeatedly flushed into the culture dish.
  • RPMI 1640 (10% FBS) medium to stop lysis, centrifuge at 400g for 3 minutes, and discard the supernatant.
  • the cells were placed in a 10 mm culture dish and cultured in RPMI 1640 (10% FBS) medium with recombinant mouse GM-CSF (20 ng/mL) added at 37 degrees Celsius and 5% CO 2 for 7 days. On the third day, shake the culture bottle gently and add the same volume of RPMI 1640 (10% FBS) medium containing GM-CSF (20ng/mL).
  • Nanoparticles loaded with whole-cell antigen derived from ⁇ cells 500 ⁇ g, including 250 ⁇ g of nanoparticles loaded with water-soluble components and 250 ⁇ g of nanoparticles loaded with non-water-soluble components) or blank nanoparticles (500 ⁇ g) + free lysate and BMDC (10 million) were incubated in 15mL RPMI1640 complete medium for a total of 96 hours (37°C, 5% CO 2 ); 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).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the incubated DCs were collected by centrifugation at 400g for 5 min, and then the cells were washed twice with PBS, resuspended in PBS and sonicated at 7.5W for 20 min.
  • the sample was then centrifuged at 2000g for 20 minutes and the supernatant was collected.
  • the supernatant was centrifuged at 7000g for 20 minutes.
  • the supernatant was collected and filtered using a 0.22 ⁇ m filter membrane.
  • the filtrate was collected after extrusion.
  • the filtrate was then centrifuged at 15000g for 120 minutes. Collect and discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain a nanovaccine based on the DC cell membrane.
  • the particle size of the nanovaccine is 120 nanometers.
  • the nanovaccine prepared by using DCs activated by nanoparticles loaded with whole cell antigens is Nanovaccine 1; the nanovaccine prepared by using DCs activated by blank nanoparticles + free lysate is Nanovaccine 2.
  • step (4) collect the activated DCs in step (4) and centrifuge them at 400g for 5 minutes, then wash the cells twice with PBS, resuspend the cells in PBS and use them directly as DC vaccine 3.
  • Example 2 Antigen-presenting cells prepare nanovaccine for prevention of type I diabetes
  • the precipitation part can convert the non-water-soluble components in NIT-1 ⁇ cells that are insoluble in pure water into soluble in aqueous solution.
  • the above obtained water-soluble components derived from cell lysates and the original non-water-soluble components dissolved in sodium deoxycholate are mixed at a mass ratio of 1:1, which is the antigen component for preparing particles that activate antigen-presenting cells. .
  • the particle size of the blank microparticles is about 1.4 ⁇ m.
  • the preparation materials and preparation methods of the blank microparticles are the same. They load equal amounts of rapamycin and tacrolimus but do not load any cleaved water-soluble components and non-water-soluble components.
  • the preparation method of microparticles loaded with several type I diabetes antigen polypeptides is the same.
  • the polypeptides loaded with equal masses are Insulin B 9-23, InsulinA 14-20, IGRP 206-214, and GAD 225-244.
  • the average particle size is about 1.5 ⁇ m, and the surface potential of the micron vaccine is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with approximately 90 ⁇ g of polypeptide components and an equal amount of immunosuppressive agents.
  • the cells after incubation were collected and labeled with live-dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody and FOXP3 antibody, and then flow cytometry was used to analyze the proportion of CD4 + CD25 + FOXP3 + T cells in the T cell subsets.
  • the ⁇ -cell whole-cell antigen loaded on the micron particles can be degraded into antigenic epitopes after being engulfed by the antigen-presenting cell B cells and presented to the surface of the antigen-presenting cell.
  • T cells that can recognize the ⁇ -cell whole-cell antigen are called It can recognize ⁇ -cell whole cell antigen epitopes and be activated and highly express specific surface markers.
  • T cells that highly express specific markers analyzed by flow cytometry are regulatory T cells, that is, T regs that can inhibit effector T cells (T eff ) that recognize and kill ⁇ cells.
  • the nanovaccine according to the present invention has a preventive effect on type I diabetes.
  • DC+B cells activated by micron particles loaded with ⁇ -cell whole cell antigens present a wider spectrum of ⁇ -cell antigens, so the number of T reg cell clones that can be activated by the nanovaccine prepared by it is also wider and can inhibit
  • the specific T eff types that recognize the same antigen as T reg are also broad-spectrum, and the effect of preventing type I diabetes is better.
  • the nanovaccine prepared by DC+B cells activated by micron particles loaded with whole cell antigens can induce CD8 + Ly49 + T cells and CD4 + CD25 + FOXP3 + T cells, respectively accounting for CD8 + T cells and CD4 + T cells.
  • the proportion of cells was significantly higher than that induced by nanovaccines prepared by peptide-loaded microparticles and blank microparticles + free lysate-activated DC + B cells. It can be seen that the nanovaccine prepared by the antigen-presenting cells activated by micron particles loaded with whole-cell antigens according to the present invention can better activate ⁇ -cell-specific T reg cells with the ability to recognize and inhibit T eff .
  • micron particles are prepared from a mixture of mouse pancreatic islet tissue lysate whole cell components and ⁇ -cell lysate whole cell components, and then the micron particles are used to activate antigen-presenting cells, and then the antigen-presenting cells are used to prepare micron vaccines.
  • mice After the mice were killed, the mouse islet tissue was collected. The islet tissue was passed through a cell filter to prepare a single-cell suspension. An appropriate amount of pure water was added and then frozen and thawed 5 times, accompanied by ultrasound to destroy the lysed cells. The lysate was Centrifuge at 3000g for 3 minutes and take the supernatant to form the water-soluble component. Dissolve the precipitated part with 8M urea aqueous solution (containing 500mM sodium chloride) to obtain the non-water-soluble component that has been dissolved by the dissolving agent.
  • 8M urea aqueous solution containing 500mM sodium chloride
  • 8M urea aqueous solution containing 500mM sodium chloride
  • 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.
  • the above water-soluble components derived from ⁇ -cell lysates and the water-soluble components derived from pancreatic islet tissue lysates are mixed at a mass ratio of 1:1 to form a water-soluble component mixture; the water-insoluble components derived from ⁇ -cell lysates The component and the water-insoluble component derived from the islet tissue lysate are mixed at a mass ratio of 1:1 to form a water-insoluble component mixture; the water-soluble component mixture and the water-insoluble component mixture are mixed at a mass ratio of 2: 1.
  • the mixture is a mixture of whole cell components, containing whole cell antigens related to type I diabetes.
  • the double emulsion method in the solvent evaporation method is used to prepare micron particles.
  • the molecular weight of PLA, the material used to prepare micron particles is 40KDa.
  • the immunosuppressant used is a mixed immunosuppressant of cyclosporine A and tacrolimus.
  • the substance used to increase lysosome escape is NH 4 HCO 3 , and the mixed immunosuppressant and the substance that increases lysosome escape are distributed inside the micron particles.
  • cyclosporine A, tacrolimus and PLA are together Dissolved in the organic phase, NH 4 HCO 3 is dissolved together with the lysate components in the first aqueous phase.
  • the preparation method is as described above.
  • the double emulsion method is first used to load the whole cell lysate components, NH 4 HCO 3 and immunosuppressants inside the micron particles. Then 100 mg of the micron particles are centrifuged at 6000g for 10 minutes and discarded. After the supernatant, resuspend the pellet in 10 mL of ultrapure water containing 4% trehalose and freeze-dry for 48 hours for later use; resuspend it in 9 mL of PBS before use and then add 1 mL of lysate component (protein concentration 80 mg/mL) and incubated at room temperature for 10 minutes to obtain a micron particle system loaded with whole cell lysate components both inside and outside.
  • lysate component protein concentration 80 mg/mL
  • the average particle size of the obtained micron particles is about 2.55 ⁇ m, and the surface potential of the micron vaccine is about -4mV.
  • Each 1 mg of PLA micron vaccine is loaded with approximately 130 ⁇ g of protein or peptide components.
  • Each 1 mg of PLA micron vaccine is loaded with cyclosporine A and tacrolimus. 0.025mg, loading NH 4 HCO 3 is 0.05mg.
  • This example uses DC cells and B cells derived from peripheral blood as antigen-presenting cells. After the mice were sacrificed, peripheral blood was collected, and gradient centrifugation was used to isolate mouse peripheral blood mononuclear cells (PBMC). Then flow cytometry was used to separate CD19 + B cells and CD11c + DC from mouse PBMC. The above The mixed antigen-presenting cells used are B cells and DCs mixed at a ratio of 1:1.
  • Whole-cell antigen-loaded micron particles (500 ⁇ g) were incubated with mixed antigen-presenting cells (a total of 10 million cells, including 5 million DCs and 5 million B cells) in 15 mL of high-glucose DMEM complete medium for 72 hours ( 37°C, 5% CO2), the incubation system contains cytokine combination 1: TGF- ⁇ (2000U/mL), IL-4 (200U/mL), IL-7 (1000U/mL), IL-10 (2000U/mL) mL).
  • whole-cell antigen-loaded micron particles 500 ⁇ g
  • mixed antigen-presenting cells 10 million cells in total, including 5 million DCs and 5 million B cells
  • the incubation system contains cytokine combination 2: TNF-a (2000U/mL), IL-12 (200U/mL), IL-6 (1000U/mL), IL-15 (2000U /mL).
  • cytokine combination 1 TGF- ⁇ (2000U/mL), IL-4 (200U/mL), IL-7 (1000U/mL), IL-10 (2000U/mL).
  • step (4) Collect the mixed antigen-presenting cells activated in step (4) (10 million cells, including 5 million DCs and 5 million B cells) by centrifugation at 300 g for 4 minutes, and then use 4°C phosphate buffer containing protease inhibitors The cells were washed twice with PBS solution, resuspended in PBS water and sonicated at low power (5W) for 30 minutes. The sample was then filtered through membranes with pore sizes of 10 ⁇ m, 5 ⁇ m, 2 ⁇ m, 1 ⁇ m and 0.45 ⁇ m in sequence and the filtrate was collected. The filtrate was centrifuged at 16000g for 20 minutes and the precipitate was discarded to collect the supernatant.
  • step ( 2) The prepared micron particles (80 mg) were mixed, ultrasonicated at 20W for 3 minutes, incubated for 15 minutes, filtered using a 5 ⁇ m filter membrane, extruded, and the filtrate was collected. Then, the filtrate was centrifuged at 8000g for 15 minutes, and the supernatant was discarded and collected. Precipitate, resuspend the precipitate in PBS to obtain the micron vaccine.
  • step (2) Combine the supernatant with step (2) The prepared micron particles (80 mg) were mixed and ultrasonicated at 20W for 3 minutes, incubated for 15 minutes, filtered with a 5 ⁇ m filter membrane, extruded, and the filtrate was collected. The filtrate was then centrifuged at 8000 g for 15 minutes, and the supernatant was discarded to collect the precipitate. Resuspend the precipitate in PBS to obtain Micron Vaccine 4, with a particle size of 2.60 ⁇ m.
  • the particles (80 mg) were mixed, ultrasonicated at 20W for 3 minutes, incubated for 15 minutes, filtered through a 5 ⁇ m filter, extruded, and the filtrate was collected. The filtrate was then centrifuged at 8000 g for 15 minutes, and the supernatant was discarded to collect the precipitate. The precipitate was collected in After resuspension in PBS, micron vaccine 5 was obtained, with a particle size of 2.60 ⁇ m.
  • mice Select 6-8 week old female C57BL/6 mice as model mice, and subcutaneously inject 100 ⁇ g of Micron vaccine 1 or Micron vaccine into each mouse on day 0, day 7, day 14, day 28 and day 42 respectively. 2. Or Micron vaccine 3, or Micron vaccine 4 or Micron vaccine 5.
  • the mice were sacrificed on day 45, the mouse spleens were removed, and a single cell suspension of splenocytes was prepared. B cells and T cells were sorted from the mouse splenocytes using magnetic bead sorting. 100 ⁇ g of micron particles loaded with ⁇ -cell whole cell antigen, 5 million B cells, and 1 million T cells were incubated in 5 mL of RPMI1640 complete medium for 48 hours (37°C, 5% CO 2 ).
  • the cells after incubation were collected and labeled with live-dead cell dye, CD3 antibody, CD8 antibody, CD4 antibody, CD25 antibody, Ly49 antibody and FOXP3 antibody, and then flow cytometry was used to analyze the proportion of CD4 + CD25 + FOXP3 + T cells in the T cell subsets.
  • the ⁇ -cell whole-cell antigen loaded on the micron particles can be degraded into antigenic epitopes after being engulfed by the antigen-presenting cell B cells and presented to the surface of the antigen-presenting cell.
  • the particle surface is loaded with mixed antigen-presenting cell membrane components activated by whole-cell antigen-loaded particles, which can significantly improve the efficacy of the vaccine in preventing type I diabetes; moreover, the particle surface is loaded with cells containing type I diabetes-related antigens.
  • the cell membrane component can also improve the efficacy of the micron vaccine in preventing type I diabetes; moreover, the best effect in preventing type I diabetes is when the particle surface is loaded with both the mixed antigen-presenting cell cell membrane component and the antigen-containing cell membrane component.
  • Micron Vaccine 1 is significantly better than Micron Vaccine 3, indicating that activating the antigen-presenting cells using particles loaded with whole cell antigens before loading the cell membrane components of the antigen-presenting cells on the particle surface can significantly improve the efficacy of the final particles prepared. Moreover, Micron Vaccine 1 is better than Micron Vaccine 2, indicating that adding specific cytokines when using particles to activate mixed antigen-presenting cells can help improve the efficacy of vaccines prepared from the cell membranes of antigen-presenting cells. In summary, the micron vaccine of the present invention has a preventive effect on type I diabetes.
  • Micron vaccine 1 can induce more self-antigen-specific T regs than Micron vaccine 2 and Micron vaccine 3; while Micron vaccine 5 can induce more self-antigen-specific T regs than Micron vaccine 1.
  • the cell membrane loaded with whole cell antigens internally the cell membrane loaded with activated antigen-presenting cells on the surface, especially the cell membrane of mixed antigen-presenting cells, is conducive to inducing the production of more self-antigen-specific T regs . Because T eff with cell-killing ability can be inhibited, the induced T eff can be effective in preventing or treating type I diabetes.
  • This example uses a mouse type I diabetes model to illustrate how to use nano-vaccines derived from antigen-presenting cells to prevent autoimmune diseases.
  • pancreatic tissue is first lysed to prepare water-soluble antigens and water-insoluble antigens of pancreatic tissue; then, a nanoparticle system loaded with water-soluble antigens and water-insoluble antigens of pancreatic tissue is prepared.
  • siliconization and adding charged substances were used to increase the loading capacity of the antigen, and only one round of mineralization was performed.
  • nanoparticles are first used to activate antigen-presenting cells, and then the antigen-presenting cells are used to prepare nanovaccines.
  • pancreatic tissues of the mice were harvested. Cut the pancreatic tissue into pieces and then grind it. Add collagenase and incubate it in RPMI 1640 medium for 30 minutes. Then prepare a single cell suspension through a cell filter. Add an appropriate amount of pure water and freeze and thaw repeatedly 5 times, accompanied by ultrasound to destroy the lysis results. cell. After the cells are lysed, centrifuge the lysate at 5000g for 5 minutes and take the supernatant, which is the water-soluble antigen soluble in pure water; add 10% sodium dodecyl sulfate (SDS) to the resulting precipitate.
  • SDS sodium dodecyl sulfate
  • the nanoparticles and the blank nanoparticles used as a control were prepared by the solvent evaporation method, and appropriate modifications and improvements were made.
  • two modification methods, low-temperature siliconization technology and addition of charged substances, were used to increase the loading capacity of the antigen. .
  • the molecular weight of the nanoparticle preparation material PLA used is 40KDa
  • the immunosuppressant used is fingolimod
  • fingolimod and PLA are dissolved in the organic phase.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load antigens and immunosuppressants inside the nanoparticles.
  • the average particle size of the nanoparticles is about 350nm, and the surface potential of the nanoparticles is about -3mV; each 1 mg of PLA nanoparticles is loaded with approximately 260 ⁇ g of protein or peptide components, and each 1 mg of PLA nanoparticles is loaded with 0.03 mg of fingolimod.
  • the preparation materials and preparation methods of the control peptide nanoparticles are the same, and they are loaded with four beta cell peptide antigens of equal mass, and are otherwise the same as the nanoparticles loaded with whole cell antigens.
  • the peptide nanoparticles load 0.03mg of fingolimod per 1mg of PLA nanoparticles, the average particle size is about 350nm, and the surface potential of the nanoparticles is about -3mV.
  • the loaded polypeptide antigens are InsulinB 9-23, InsulinA 14-20, IGRP206-214, and GAD 225-244.
  • mice were killed, the mouse lymph nodes were removed.
  • the mouse lymph nodes were cut into pieces and filtered through a cell mesh to prepare a single cell suspension of lymph node cells.
  • CD11c + DCs were then sorted from lymph node cell single cell suspensions using flow cytometry.
  • BMDM bone marrow-derived macrophages
  • mice were anesthetized and sacrificed by dislocation.
  • the mice were disinfected with 75% ethanol.
  • a small opening was made on the back of the mouse with scissors.
  • the skin was directly torn open to the calf joint of the mouse by hand, and the foot joint and foot joint of the mouse were removed. skin.
  • Macrophage colony-stimulating factor stimulates bone marrow cells to differentiate into mononuclear macrophages at a concentration of 40ng/mL. After culturing for 8 days, the morphological changes of macrophages were observed under a light microscope. After 8 days, the cells were digested and collected, and incubated with anti-mouse F4/80 antibody and anti-mouse CD11b antibody for 30 minutes at 4°C in the dark, and flow cytometry was used to identify the proportion of successfully induced macrophages.
  • M-CSF Macrophage colony-stimulating factor
  • Nanovaccines prepared from antigen-presenting cells are used to prevent type I diabetes
  • nanoparticles loaded with ⁇ -cell whole cell antigen were prepared using PLA as the nanoparticle skeleton material and Tripterygium wilfordii as the immunosuppressant. Use nanoparticles to activate antigen-presenting cells and then present the antigen to the cell membrane to prepare nano-vaccines to prevent type I diabetes.
  • the nanoparticles were prepared using the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLA used is 30KDa
  • the immunosuppressant used is tripterygium wilfordii.
  • the preparation method is as described above.
  • the double emulsion method is first used to load NIT-1 cell whole cell antigen and tripterygium wilfordii inside the nanoparticles. Then 100 mg of nanoparticles are centrifuged at 13000g for 25 minutes, and then 10 mL of 4% seaweed is used. The sugar was resuspended in ultrapure water and freeze-dried for 48 hours before use.
  • the average particle size of the nanoparticles is about 250nm, and the surface potential of the nanoparticles is about -4mV; each 1 mg of PLA nanoparticles is loaded with approximately 90 ⁇ g of protein or peptide components, and 0.02 mg of Tripterygium wilfordii.
  • the nanoparticles were used as nanovaccine3 in mouse animal experiments.
  • the preparation method is the same as Example 1.
  • the control group in this study was the PBS group.
  • Three-week-old female NOD mice were selected for this experiment.
  • 10 NOD mice in each group were subcutaneously injected with 100 ⁇ g Nano Vaccine 1, or 100 ⁇ g Nano Vaccine 2, or 100 ⁇ g Nano Vaccine 3, or 100 ⁇ L PBS every 7 days starting from the third week, for 6 consecutive weeks.
  • the blood sugar status of mice in each group was recorded every day starting from the 8th week. Diabetes begins when blood sugar is higher than 11.0mmol ⁇ L-1. The incidence of diabetes in NOD mice at different time periods was recorded.
  • Nanoparticle prepared in the form of vesicles loaded with cell membrane components; while nanovaccine 1 is loaded with whole cell antigens internally and presents complex antigens on the surface to the cell membrane.
  • the surface of the solid sphere is loaded with cell membrane nanoparticles, which shows that the effect of solid nanovaccines loaded with whole cell antigens internally and membrane components on the surface is better than that of vesicle nanovaccines that are only loaded with antigens on the surface. Since the surface of the activated antigen-presenting cell membrane has a complex of MHC molecules bound to antigen polypeptides, the surfaces of both Nano Vaccine 1 and Nano Vaccine 2 are loaded with complexes of MHC molecules bound to antigen polypeptides.
  • Nanovaccine 1 with a membrane structure can be directly T cells are induced into regulatory T cells (T reg ) with negative regulatory functions. They can also be phagocytosed by antigen-presenting cells and the loaded antigens are degraded and presented by the antigen-presenting cells and then indirectly activate regulatory T cells (T reg) . ).
  • T reg regulatory T cells
  • the nanovaccine according to the present invention has a good preventive effect on type I diabetes.
  • 8M urea was first used to lyse the islet tissue and dissolve the islet tissue lysate components. Then, PLA is used as the nanoparticle skeleton material and cyclophosphamide is used as the immunosuppressant to prepare nanoparticles loaded with whole cell antigens. The nanoparticles are used to activate the antigen-presenting cells and then the antigen-presenting cells are used to prepare nano-vaccines for preventing autoimmunity. Disease - Type I Diabetes.
  • nanoparticles were prepared by solvent evaporation method.
  • the molecular weight of the PLA material used to prepare the nanoparticles is 20KDa
  • the immunosuppressant used is cyclophosphamide
  • the lysate components and cyclophosphamide are contained inside the nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to load the lysate components and immunosuppressants inside the nanoparticles. After loading the antigen lysis components and immunosuppressants inside, 100 mg of nanoparticles are centrifuged at 12000g.
  • each 1 mg of PLA nanoparticles is loaded with approximately 100 ⁇ g of protein or peptide components, and each 1 mg of PLA nanoparticles is loaded with 0.04 mg of cyclophosphamide.
  • the preparation materials and methods of blank nanoparticles are the same, and the particle size is about 280 nm.
  • the blank nanoparticles are loaded with an equal amount of immune preparations but do not load any lysate components.
  • control nanoparticles were loaded with four antigen peptides of equal mass (Insulin B 9-23, Insulin A 14-20, IGRP 206-214, and GAD 225-244) to replace the lysate components.
  • the other nanoparticles were loaded with whole cell antigens.
  • the particles are the same.
  • the control nanoparticles carry 0.04mg of cyclophosphamide per 1mg of PLA nanoparticles, the particle size is about 280nm, the surface potential is about -3mV, and each 1mg of PLA nanoparticles carries about 100 ⁇ g of peptide components.
  • mice were sacrificed, the mouse lymph nodes were removed, and a mouse lymph node single cell suspension was prepared, and then flow cytometry was used to sort out CD11c + DC and CD19 + B cells from the lymph node cell single cell suspension.
  • Whole-cell antigen-loaded nanoparticles (500 ⁇ g) or peptide nanoparticles (500 ⁇ g) were incubated with DCs (5 million cells) and B cells (5 million cells) in 20 mL of high-glucose DMEM complete medium for 72 hours (37°C, 5 % CO 2 ), or incubate whole-cell antigen-loaded nanoparticles (500 ⁇ g) and DCs (10 million) in 20 mL high-glucose DMEM complete medium for 72 hours (37°C, 5% CO 2 ); the incubation system contains GM-CSF (1000U/mL), IL-2 (100U/mL), IL-10 (2000U/mL), TGF- ⁇ (2000U/mL).
  • Cell nanoparticles (20 mg) were mixed, stirred at 2000 RPM for 2 minutes, filtered through a 0.45 ⁇ m filter, extruded, and the filtrate was collected. Then, the filtrate was centrifuged at 12,000 g for 30 minutes. The supernatant was discarded to collect the precipitate, and the precipitate was placed in PBS. After resuspension, the nano vaccine is obtained.
  • the particle size of the nano vaccine is 300 nanometers.
  • nanovaccine of the present invention has a good preventive effect on type I diabetes. Moreover, the preventive effect of nanovaccines prepared from DCs and B cells activated by nanoparticles loaded with whole cell antigens was better than that of nanovaccines prepared from DCs and B cells activated by nanoparticles loaded with four antigen peptides.
  • Example 7 Nanovaccine prepared from activated antigen-presenting cells for the treatment of type I diabetes
  • This example uses type I diabetes as a model to illustrate how to use antigen-presenting cells activated by nanoparticles to prepare nano-vaccines and use such vaccines to prevent and treat autoimmune diseases.
  • ⁇ cells NIT-1 cells
  • PLA was used as the nanoparticle skeleton material
  • rapamycin and Gusperimus were used as immunosuppressants to prepare nanoparticles.
  • the nanoparticles were used to activate antigen-presenting cells in vitro to prepare nanovaccines to treat type I diabetes.
  • the nanoparticles were prepared by solvent evaporation method.
  • the molecular weight of PLA, the material used to prepare the nanoparticles, is 20KDa.
  • the immunosuppressants used are rapamycin and guanilimus, and the immunosuppressants are distributed inside the nanoparticles.
  • the preparation method is as mentioned above.
  • the lysate mixture and immunosuppressant are first loaded inside the nanoparticles using the double emulsion method. After loading the lysate and adjuvant inside, 100 mg of the nanoparticles are centrifuged at 10,000g for 20 minutes and used Resuspend 10 mL of ultrapure water containing 4% trehalose and freeze-dry for 48 h.
  • each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components, and each 1 mg of PLGA nanoparticles contains 0.03 mg of rapamycin and guanilimus each. .
  • PBMC Peripheral blood mononuclear cells
  • CD11c + DCs were sorted from PBMC using flow cytometry.
  • DC and BMDM were used simultaneously as antigen-presenting cells.
  • the preparation method of BMDM is the same as in Example 4.
  • mice with onset of diabetes were selected for this experiment.
  • the blood glucose of mice in the PBS control group was always higher than normal.
  • Some mice in the nanovaccine treatment group recovered from type I diabetes.
  • the effect of nanovaccines prepared by mixing activated DCs and macrophages is better than that of nanovaccines prepared by DCs activated by nanoparticles, indicating that nanovaccines prepared by a variety of antigen-presenting cells activated by nanoparticles are more effective.
  • the nanovaccine prepared by the antigen-presenting cells of the present invention has a good therapeutic effect on type I diabetes.
  • This example uses type I diabetes as a model to illustrate how to use nanoparticles loaded with ⁇ -cell whole cell antigen to activate antigen-presenting cells, and then use nanovaccines prepared from antigen-presenting cells to treat type I diabetes.
  • the nanoparticles were prepared using the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa, and the inhibitors used are rapamycin and everolimus.
  • 100 mg of the nanoparticles were centrifuged at 10,000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use.
  • the average particle size of the nanoparticles is about 360nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 ⁇ g of rapamycin and everolimus each. mg.
  • the preparation materials and preparation methods of blank nanoparticles are the same as above.
  • the particle size is about 350 nm. They only load an equal amount of adjuvant but do not load any lysate components.
  • the blood sugar of mice treated with nanovaccines prepared by PBS control and blank nanoparticle-activated antigen-presenting cells was always higher than normal.
  • the nanovaccine treatment group prepared by nanoparticles loaded with whole-cell antigens and activated antigen-presenting cells recovered from type I diabetes in some mice.
  • nanovaccines with CD32 monoclonal antibody targets are more effective than nanovaccines without targets.
  • the nanovaccine prepared by the antigen-presenting cells of the present invention has a good therapeutic effect on type I diabetes.
  • the nanovaccine uses CD32 monoclonal antibody as the active target.
  • any target with the ability to target target cells such as mannose, mannan, CD205 monoclonal antibody, CD19 monoclonal antibody, etc., can also be used. head.
  • Example 9 Antigen-presenting cell-based nanovaccine for the prevention of type I diabetes
  • This example uses mouse pancreatic tissue to illustrate how to prepare micron particles loaded with ⁇ -cell pancreatic tissue whole cell antigens, then use the micron particles to activate antigen-presenting cells, prepare the antigen-presenting cells into nano-vaccines, and apply this Nanovaccine prevents type I diabetes.
  • the pancreas contains mouse beta cells and can be used as a source of islet beta cells to prepare vaccines. First, the mouse pancreatic tissue was extracted and the water-soluble and non-water-soluble components of the whole cells of the tissue were prepared.
  • the organic polymer material PLGA was used as the nanoparticle skeleton material, and the mRNA encoding TGF- ⁇ and rapamycin were used as immunosuppressants to prepare micron particles loaded with whole cell antigens using a solvent evaporation method, and the antigens activated by the micron particles were Presenting cells to prepare nanovaccines.
  • mice were sacrificed and their pancreatic tissues were harvested. Cut the pancreatic tissue into pieces and then grind it. Add an appropriate amount of pure water through a cell strainer and repeat freezing, thawing and sonication at least 8 times. After the tissue cells are lysed, irradiate the tissue cell lysate with ultraviolet light for 15 minutes, heat the lysate at 95°C for 10 minutes, and then centrifuge the tissue lysate at a speed greater than 3000 RPM for 5 minutes. The supernatant is the tissue cell.
  • Water-soluble components that are soluble in pure water add 8M urea aqueous solution to the resulting precipitate to dissolve the precipitate, and the original non-water-soluble components that are insoluble in pure water can be converted into soluble in 8M urea aqueous solution.
  • the double emulsion method in the solvent evaporation method is used to prepare micron particles.
  • the molecular weight of the micron particle preparation material PLGA used is 24KDa-38KDa.
  • the immunosuppressant used is mRNA encoding TGF- ⁇ and rapamycin.
  • the mRNA encoding TGF- ⁇ and rapamycin are distributed inside the micron particles.
  • the mRNA is dissolved in the aqueous phase and rapamycin is dissolved in the organic phase.
  • the preparation method is as mentioned above.
  • the average particle size of the microparticles is about 1.5 ⁇ m, and the surface potential Zeta potential is about -8mV.
  • Each 1 mg PLGA micron particle is loaded with approximately 90 ⁇ g of protein or peptide components, and each 1 mg PLGA micron particle is loaded with 0.01 mg of mRNA.
  • the particle size of the blank microparticles is about 1.5 ⁇ m. The blank microparticles carry the same amount of mRNA but do not load any lysate components.
  • the preparation of BMDC is the same as in Example 1.
  • the preparation method of B cells derived from mouse peripheral blood is the same as above.
  • This example uses type I diabetes as a model to illustrate how to use nanoparticles loaded with ⁇ -cell whole cell antigen to activate antigen-presenting cells, and then use the nanovaccine prepared by the antigen-presenting cells to prevent type I diabetes. Moreover, proper treatment of ⁇ -cells before lysis can increase the antigen content, thereby improving the efficiency of antigen-loaded particles in activating antigen-presenting cells.
  • Sodium aqueous solution dissolves the precipitated part to convert the non-water-soluble components in NIT-1 cells that are insoluble in pure water into soluble in 8M urea aqueous solution.
  • Mixing the above water-soluble components and non-water-soluble components at a mass ratio of 1:1 is the antigen source for preparing control nanoparticles.
  • the average particle size of the nanoparticles is about 260nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 ⁇ g of rapamycin and everolimus each. mg.
  • DC is a mixture of DC derived from peripheral blood and BMDC.
  • the preparation methods of both are the same as above.
  • peripheral blood-derived DCs 8 million and BMDC (8 million) were collected by centrifugation at 400g for 5 minutes, and then the cells were washed twice with 4°C phosphate buffer solution (PBS) containing protease inhibitors.
  • PBS 4°C phosphate buffer solution
  • the cells were resuspended in PBS water and ultrasonicated at 4°C for 1 minute at low power (20W) and treated with a homogenizer at 1000 rpm for 3 minutes.
  • the sample was then centrifuged at 3000g for 15 minutes and the supernatant was collected.
  • the supernatant was centrifuged at 8000g for 15 minutes and the supernatant was collected.
  • the supernatant was filtered and extruded using a 0.22 ⁇ m filter membrane and the filtrate was collected.
  • the filtrate was mixed with The corresponding nanoparticles (30 mg) and DSPE-PEG-CD32 monoclonal antibody (30 ⁇ g) prepared in step 2 in Example 1 were stirred at 1200 RPM for 2 minutes, filtered with a 0.45 ⁇ m filter, extruded, and the filtrate was collected, and then the filtrate was After centrifugation at 15,000 g for 30 minutes, collect and discard the supernatant to collect the precipitate. Resuspend the precipitate in PBS to obtain the nano vaccine.
  • the particle size of the nano vaccine is 280 nanometers.
  • nanovaccines prepared by antigen-presenting cells activated by nanoparticles loaded with whole cell antigens and suffering from type I diabetes was significantly reduced.
  • nanovaccines prepared using nanoparticle-activated antigen-presenting cells prepared from beta cells treated with high sugar and added chemicals were more effective than nanoparticle-activated antigen-presenting cells prepared from beta cells treated as above. of nano vaccines. It can be seen that the nanovaccine of the present invention has a preventive effect on type I diabetes, and appropriate treatment of ⁇ cells to increase the antigen content can help improve the efficacy of the nanovaccine prepared by activated antigen-presenting cells.
  • Example 11 Nanovaccine prepared by activated antigen-presenting cells for preventing type I diabetes
  • the average particle size of the nanoparticles is about 250nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 ⁇ g of rapamycin and tacrolimus each. mg.
  • DC is a mixture of DC derived from peripheral blood and BMDC.
  • the preparation methods of both are the same as above.
  • Whole-cell antigen-loaded nanoparticles (800 ⁇ g) were incubated with peripheral blood-derived DCs (10 million) and BMDC (10 million) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2).
  • the incubation system contains IL-13 (500U/mL), IL-10 (2000U/mL), IL-4 (1000U/mL) and TGF- ⁇ (2000U/mL).
  • the sample was then sequentially filtered through membranes with pore sizes of 30 ⁇ m, 10 ⁇ m, 5 ⁇ m, 2 ⁇ m, and 0.45 ⁇ m.
  • the filtrate was centrifuged at 12,000g for 25 minutes and the supernatant was discarded to collect the precipitate.
  • the precipitate was placed in physiological saline containing 4% mannitol. After medium-resuspension and freeze-drying, the nano-vaccine is obtained.
  • the particle size of the nano-vaccine is 260 nanometers.
  • nanovaccines prepared by antigen-presenting cells activated by nanoparticles loaded with whole cell antigens and suffering from type I diabetes was significantly reduced.
  • the effect of nanovaccines prepared by adding cytokines or antibodies to the antigen-presenting cells activated by nanoparticles is better than that of nanoparticle-activated antigen-presenting cells without adding any cytokines or antibodies.
  • Nanovaccine prepared from antigen-presenting cells It can be seen that the nanovaccine of the present invention has a preventive effect on type I diabetes, and the addition of cytokines and/or antibodies during the activation of antigen-presenting cells helps to improve the efficiency of preparation of activated antigen-presenting cells. Efficacy of nanovaccines.
  • the nanoparticles and control nanoparticles were prepared using the double emulsion method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 24KDa-38KDa
  • the inhibitors used are rapamycin and interleukin 2
  • KALA polypeptide WEAKLAKALAKALAKHLAKALAKALKACEA
  • 100 mg of nanoparticles were centrifuged at 12,000g for 20 minutes, resuspended in 10 mL of ultrapure water containing 4% trehalose, and freeze-dried for 48 hours before use.
  • the average particle size of the nanoparticles is about 250nm, and the surface potential of the nanoparticles is about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with 0.02 mg each of rapamycin and interleukin-2. Loaded with KALA peptide 0.03mg.
  • control nanoparticles are the same, with an average particle size of about 250nm and a surface potential of about -5mV; each 1 mg of PLGA nanoparticles is loaded with approximately 80 ⁇ g of protein and peptide components, and each 1 mg of PLGA nanoparticles is loaded with rapamycin and interleukin. 2 0.03mg each, does not load any substances that increase lysosomal escape.
  • DC is a mixture of DC derived from peripheral blood and BMDC.
  • the preparation methods of both are the same as above.
  • Whole-cell antigen-loaded nanoparticles (800 ⁇ g) were incubated with peripheral blood-derived DCs (8 million) and BMDC (8 million) in 15 mL high-glucose DMEM complete medium for 48 hours (37°C, 5% CO2).
  • the incubation system contains IL-13 (500U/mL), IL-10 (2000U/mL), IL-4 (1000U/mL) and TGF- ⁇ (2000U/mL).
  • the KALA polypeptide added in this example can also be loaded with one or more other substances that have the function of increasing lysosomal escape during actual use, such as arginine, polyarginine, and lysine. , polylysine, histidine, polyhistidine, NH 4 HCO 3 , positively charged peptides, protamine, histones, etc.
  • the nanoparticles are prepared by a solvent evaporation method.
  • the molecular weight of the nanoparticle preparation material PLGA used is 38KDa-54KDa.
  • the immunosuppressive agents used are rapamycin and mycophenolate mofetil, which increase lysosomal escape.
  • the substances are polyarginine and RALA polypeptide, and the immunosuppressant, polyarginine and RALA polypeptide are all loaded in the nanoparticles.
  • the preparation method is as mentioned above.
  • the double emulsion method is first used to cleave the components, immunosuppressants, polyarginine and RALA peptides inside the nanoparticles, and then 100mg PLGA nanoparticles are centrifuged at 13000g for 20 minutes and the precipitate is used Resuspend in 4% trehalose and freeze-dry for 48 hours before use.
  • the average particle size of the nanoparticles is about 260nm; each 1 mg of PLGA nanoparticles is loaded with approximately 140 ⁇ g of protein or peptide components, 0.03 mg each of rapamycin and mycophenolate mofetil, and 0.02 mg each of polyarginine and RALA peptide.
  • the preparation materials and methods of blank nanoparticles are the same.
  • the average particle size of blank nanoparticles is about 260nm.
  • Each 1 mg of PLGA blank nanoparticles is loaded with 0.03 mg of rapamycin and mycophenolate mofetil, and 0.02 mg of polyarginine and RALA peptide are loaded. mg, but does not load any lysate components.
  • This example uses DC2.4 cells, B cells derived from splenocytes and BMDM as mixed antigen-presenting cells.
  • the preparation methods of the three antigen-presenting cells are the same as above.
  • 1 mg of whole-cell antigen-loaded nanoparticles were incubated with 30 million mixed antigen-presenting cells (10 million DCs + 10 million B cells + 10 million BMDM) in 25 mL of high-glucose DMEM complete medium for 48 hours (37°C , 5% CO 2 ), the incubation system contains IL-7 (100U/mL), IL-10 (1000U/mL), IL-4 (500U/mL) and TGF- ⁇ (1000U/mL).
  • the activated mixed antigen-presenting cells serve as live cell vaccines3.
  • PBS phosphate buffer solution
  • the membrane was filtered and extruded, then centrifuged at 13000g for 25 minutes and resuspended in 10 mL of lyophilizing protectant (containing 2% trehalose + 2% mannitol + 1% sucrose) aqueous solution, and then freeze-dried for 48 hours to obtain the nano vaccine.
  • lyophilizing protectant containing 2% trehalose + 2% mannitol + 1% sucrose
  • the vaccine is NanoVaccine 2, with a particle size of 270 nanometers.
  • the control group in this study was the PBS group.
  • Three-week-old female NOD mice were selected for this experiment.
  • 10 NOD mice in each group were subcutaneously injected with 100 ⁇ g of nano vaccine 1, or 100 ⁇ g of nano vaccine 2, or 3 million mixed antigen-presenting cell vaccine 3 (1 million DCs) every 7 days starting from the third week.
  • +1 million B cells +1 million BMDM or 100 ⁇ L PBS, for 6 consecutive weeks.
  • the blood sugar status of mice in each group was recorded every day starting from the 8th week. Diabetes begins when blood sugar is higher than 11.0mmol ⁇ L-1. The incidence of diabetes in NOD mice at different time periods was recorded.
  • mice Select 6-8 week old female C57BL/6 mice as model mice, and subcutaneously inject 100 ⁇ g of nano vaccine 1 or nano vaccine into each mouse on days 0, 7, 14, 28 and 42. 2. Or 3 million mixed antigen-presenting cell vaccines 3 (1 million DC+1 million B cells+1 million BMDM), or PBS. The mice were sacrificed on day 45, the mouse spleens were removed, and a single cell suspension of splenocytes was prepared. B cells and T cells were sorted from the mouse splenocytes using magnetic bead sorting.
  • nano vaccine 1 is significantly better than that of nano vaccine 2 and live cell vaccine 3.
  • nanovaccine that is loaded with whole-cell antigens internally and has antigen-containing cells and activated antigen-presenting cells mixed cell membrane components loaded on the surface is better than a nanovaccine that is not loaded with antigens but only has mixed cell membrane components loaded on the surface. It also shows that only the mixed cell membrane components are loaded on the surface.
  • Nanovaccines loaded with immunosuppressants and lysosomal escape substances, as well as with antigen-containing cells and activated antigen-presenting cells mixed with cell membrane components on the surface, are also better than mixed antigen-presenting nanoparticles activated with whole-cell antigen-loaded nanoparticles. Live cell vaccine. To sum up, it is shown that the nanovaccine of the present invention, which is internally loaded with whole cell antigens and whose surface is loaded with mixed cell membrane components, has a good preventive effect on type I diabetes.
  • the surface of the nanovaccine is loaded with cell membrane components of cells containing self-antigens.
  • the membrane components of extracellular vesicles secreted by cells containing self-antigens can also be used.
  • the double emulsion method was used to prepare the micron particle system and the control micron particles.
  • the molecular weight of the micron particle skeleton material PLGA was 38KDa-54KDa.
  • the immunosuppressant used was methylprednisolone.
  • the lysosomal escape method was used. Adding substances are NH 4 HCO 3 and melittin.
  • micron particles internally loaded with lysate components, methylprednisolone, NH 4 HCO 3 and melittin, then centrifuge 100 mg of micron particles at 9000g for 20 minutes, and use 10 mL containing 4% Resuspend trehalose in ultrapure water and dry for 48 hours before use.
  • the average particle size of the micron particles is about 3.1 ⁇ m, and the surface potential is about -7mV; each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or peptide components, including 0.01 mg of methylprednisolone and 0.02 mg of NH4HCO3 and melittin. mg.
  • control micron particles The preparation materials and methods of the control micron particles are the same as the above.
  • the average particle size of the control micron particles is about 3.1 ⁇ m, and the surface potential is about -7mV.
  • Each 1 mg of PLGA micron particles is loaded with approximately 110 ⁇ g of protein or peptide components and does not load any immunosuppression. agent, only loading 0.02mg each of NH 4 HCO 3 and melittin.

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Abstract

一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法及其应用，制备方法包括以下步骤：将抗原提呈细胞与负载自身免疫疾病全细胞抗原的第一递送粒子共孵育，得到预激活的抗原提呈细胞；将预激活的抗原提呈细胞细胞膜制备成纳米囊泡或细胞膜组分负载在粒子表面的纳米粒子或微米粒子，得到自身免疫疾病疫苗。所述方法在实现源于树突状细胞的疫苗负载广谱多样的癌细胞抗原的同时克服了树突状细胞活性保持困难、无法冻干长期储存等活细胞疫苗的难题，能够制备负载广谱自身免疫疾病抗原表位的疫苗，用于自身免疫疾病的预防和治疗。

Description

一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法及其应用 技术领域
本发明涉及免疫治疗领域，尤其涉及一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法及其应用。
背景技术
自身免疫性疾病是一类严重威胁人类健康的疾病，其发病机制是人体免疫系统因为某些原因将自身的物质误认为外来抗原并发动对含有这类抗原的细胞或组织的攻击，导致一系列后果和疾病。以I型糖尿病为例，因为某些未完全研究清楚的原因，机体免疫系统误将胰岛或β细胞中的某些成分误认为外来抗原，从而启动对β细胞的攻击，并进而杀伤大部分β细胞。由于β细胞是人体内分泌胰岛素的物质，而胰岛素是控制血糖的关键物质，当大部分β细胞被杀灭后，人体就无法产生足够的胰岛素控制血糖，因而I型糖尿病就发生了。因此，如果能够预防或解除人体免疫系统对β细胞的攻击，就能预防或逆转治疗I型糖尿病。而能够诱导机体产生免疫耐受的疫苗是实现上述目的主要方法之一。由于β细胞主要是由效应性T细胞(T eff)攻击的，所以诱导能够和效应性T细胞识别相同抗原但是对效应性T细胞(T eff)具有抑制作用的调节性T细胞(T reg)就至关重要。因此，本发明旨在寻找一种高效诱导产生调节性T细胞的方法，从而找到一种能够有效预防和治疗自身免疫疾病的方法。
发明内容
为解决上述技术问题，本发明提供了一种来源于被纳米粒子和/或微米粒子激活的抗原提呈细胞，并用抗原提呈细胞的膜组分制备了一种纳米疫苗(Nanovaccine,NP)或微米疫苗(Microvaccine,MP)，可用于自身免疫疾病的预防或治疗。
本发明提供了一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法，包括以下步骤：
S1、将抗原提呈细胞与负载自身免疫疾病全细胞抗原的第一递送粒子共孵育，得到预激活的抗原提呈细胞；
S2、将预激活的抗原提呈细胞细胞膜制备成纳米囊泡，得到自身免疫疾病疫苗；
或将预激活的抗原提呈细胞的细胞膜组分负载在负载自身免疫疾病全细胞抗原的第二递送粒子上，得到自身免疫疾病疫苗；
其中，
第一递送粒子或第二递送粒子分别独立地为纳米粒子或微米粒子；
所述自身免疫疾病全细胞抗原由以下步骤制备得到：将含有自身免疫疾病抗原的细胞或组织冷冻，加水进行冻融裂解，收集上清液以及沉淀中经溶解剂溶解后转为可溶的部分，得到所述自身免疫疾病全细胞抗原；或将含有自身免疫疾病抗原的细胞或组织加溶解剂进行裂解，收集可溶部分，得到所述自身免疫疾病全细胞抗原。
进一步地，溶解剂选自尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质 降解酶、白蛋白、卵磷脂、无机盐、Triton、吐温、氨基酸、糖苷和胆碱中的一种或多种。
进一步地，将预激活的抗原提呈细胞的细胞膜组分负载在负载自身免疫疾病全细胞抗原的第二递送粒子上的同时，还包括将含有自身免疫疾病抗原细胞的细胞膜组分负载在第二递送粒子上。其中，细胞膜组分包括细胞膜和/或细胞外囊泡膜。
进一步地，当递送粒子(以下“递送粒子”均表示第一递送粒子或第二递送粒子)同时负载抗原提呈细胞的膜组分和含有自身免疫疾病抗原的细胞的膜组分(以下均以β细胞的膜组分为例进行说明)时，具体步骤包括：
(1)将一种或多种抗原提呈细胞与负载自身免疫疾病全细胞抗原的纳米粒子和/或微米粒子共孵育一定时间以激活抗原提呈细胞；
(2)将激活的抗原提呈细胞进行机械破坏、膜过滤、梯度离心或化学处理，得到来源于抗原提呈细胞的细胞膜碎片和/或纳米囊泡；
(3)将含有自身免疫疾病抗原的细胞(如β细胞)进行机械破坏、膜过滤、梯度离心或化学处理，得到来源于β细胞的细胞膜碎片和/或纳米囊泡；
(4)将步骤(2)和(3)得到的产物与负载自身免疫疾病全细胞抗原的第二递送粒子共作用，即得。
进一步地，机械破坏方式选自超声、均质化、匀浆、高速搅拌、高压破坏、高剪切力破坏、溶胀、皱缩中的一种或多种。
进一步地，共作用方式选自共孵育、超声、共挤出、超滤、透析、搅拌、均质化和匀浆中的一种或多种。
进一步地，在步骤S1中，共孵育时孵育体系中含有细胞因子和/或抗体；所述细胞因子选自白介素1受体拮抗剂、生长因子、白介素、干扰素、肿瘤坏死因子、集落刺激因子、活化素和抑制素中的一种或多种；所述抗体选自PD1抗体、PD-L1抗体、CTAL-4抗体、TIGIT抗体、TIM-3抗体、LAG-3抗体、αCD-8抗体、αCD-28抗体、αCD-40抗体、αOX-40抗体和αOX-40L抗体中的一种或多种。
进一步地，细胞因子包括但不限于白介素1受体拮抗剂(IL-1ra)、白介素2(IL-2)、转化生长因子-β(TGF-β)、白介素7(IL-7)、白介素10(IL-10)、白介素14(IL-14)、白介素4(IL-4)、白介素13(IL-13)、白介素15(IL-15)、白介素21(IL-21)、白介素17(IL-17)、白介素12(IL-12)、白介素6(IL-6)、白介素33(IL-33)、γ干扰素(IFN-γ)、TNF-α、粒细胞－巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)、活化素(activins)、抑制素(inhibins)。
优选地，孵育体系中包括以下组合中的一种：
(1)GM-CSF、IL-2、IL-7、IL-10和CD40抗体；
(2)TGF-β、IL-4、IL-7、IL-10(最优选)；
(3)GM-CSF、IL-2、IL-4、IL-10和PD-L1抗体(PD-L1抗体为可选物质)；
(4)TGF-β、IL-10、IL-4和PD-L1抗体；
(5)GM-CSF、IL-2、IL-10和TGF-β；
(6)IL-2、IL-10、IL-4和TGF-β；
(7)IL-10、IL-2、IL-4和IL-13；
(8)IL-13、IL-10、IL-4和TGF-β；
(9)GM-CSF、IL-2、IL-10和IL-13。
进一步地，在步骤S1中，共孵育时孵育体系中含有葡萄糖和/或毒胡萝卜素。
进一步地，抗原提呈细胞(APC)选自树突状细胞(Dendritic cell,DC)、B细胞和巨噬细胞中的至少一种。
本发明所述的自身免疫疾病包括但不限于I型糖尿病、类风湿性关节炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎、系统性红斑狼疮、系统性血管炎、硬皮病、溃疡性结肠炎、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病。
本发明所述的纳米疫苗或微米疫苗，其特征在于：所述自身免疫疾病为I型糖尿病时，含有与I型糖尿病相关抗原的细胞为β细胞；所述组织为含有β细胞的组织，如胰腺组织和/或胰岛组织。
进一步地，被激活的抗原提呈细胞制备纳米疫苗之前可以进行适当洗涤，洗涤过称中使用的洗涤液中含有蛋白酶抑制剂。
进一步地，制备纳米疫苗或微米疫苗的超声处理抗原提呈细胞的过程为低功率超声(低于500W)；制备纳米疫苗或微米疫苗的梯度离心为离心速度依次增加的梯度离心；制备纳米疫苗或微米疫苗的膜过滤时所使用的滤膜的孔径依次由大到小；共作用时间大于20秒。
进一步地，纳米疫苗粒径大于30nm小于1000nm；微米疫苗粒径大于1μm小于50μm。
进一步地，用于激活抗原提呈细胞的纳米粒子粒径范围在10nm-1000nm,用于激活抗原提呈细胞的微米粒子粒径范围在1μm到50μm。
进一步地，第一递送粒子或第二递送粒子的内部和/或表面负载有抑制免疫的物质。
进一步地，抑制免疫的物质选自微生物来源的免疫抑制剂、人或动物免疫系统的产物、mRNA、DNA、固有免疫抑制剂、适应性免疫抑制剂、化学合成药物、真菌多糖类和中药类中的一种或多种；还可以选自糖皮质激素类药、钙调磷酸酶抑制药、抗代谢药、抗体、细胞因子、烷化剂、植物药成分和矿物药成分中的一种或多种；还可以选自环孢素、雷帕霉素、他克莫司、胍立莫司、芬戈莫德、甲基强的松龙、雷公藤、霉酚酸酯、环磷酰胺、硫唑嘌呤、依维莫司、山地明、赛斯平、环孢多肽A、环孢灵、新出地明、抗IL-2受体单克隆抗体、TGF-β、白介素、人参和黄芪中的一种或多种。
进一步地，第一递送粒子或第二递送粒子上负载有带正电荷的多肽(如KALA多肽、RALA多肽、蜂毒肽等)、精氨酸、聚精氨酸、赖氨酸、聚赖氨酸、组氨酸、聚组氨酸、 NH 4HCO 3、鱼精蛋白和组蛋白中的一种或多种。
进一步地，纳米疫苗或微米疫苗的表面连接有主动靶向功能的靶头，靶头可为甘露糖、甘露聚糖、CD19抗体、CD20抗体、BCMA抗体、CD32抗体、CD11c抗体、CD103抗体、CD44抗体等。
本发明还提供一种上述自身免疫疾病疫苗在制备用于治疗或预防自身免疫疾病药物中的应用。
进一步地，抗原提呈细胞来源于自体、同种异体、细胞系或者干细胞分化中的一种或多种。
进一步地，全细胞抗原负载于纳米粒子或微米粒子表面的方式包括吸附、共价连接、电荷相互作用、疏水相互作用、一步或多步的固化、矿化和包裹中的至少一种。
进一步地，纳米粒子或微米粒子由有机合成高分子材料、天然高分子材料或无机材料制备得到，可以采用已有的制备方法制备得到，包括但不仅限于常见的溶剂挥发法、透析法、微流控法、挤出法、热熔法。
进一步地，有机合成高分子材料包括但不限于聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚乙醇酸(PGA)、聚二醇(PEG)、聚己内酯(PCL)、Poloxamer、聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)、聚乙烯亚胺(PEI)、聚三亚甲基碳酸酯(PTMC)、聚酸酐、PDON、PPDO、聚甲基丙烯酸甲酯(PMMA)、聚氨基酸、合成多肽等；天然高分子材料包括卵磷脂、胆固醇、海藻酸盐、白蛋白、胶原蛋白、明胶、细胞膜成分、淀粉、糖类、多肽等；无机材料包括三氧化二铁、四氧化三铁、碳酸盐、磷酸盐等。
进一步地，纳米粒子或微米粒子在制备过程中可以不做修饰处理，也可以采用适当的修饰技术以提高纳米粒子或微米粒子的抗原负载量。修饰技术包括但不限于生物矿化(如硅化、钙化、镁化)、凝胶化、交联、化学修饰、添加带电物质等。
进一步地，抗原被负载于纳米粒子或微米粒子表面的方式包括但不限于吸附、共价连接、电荷相互作用(如添加带正电的物质、添加带负电的物质)、疏水相互作用、一步或多步的固化、矿化、包裹等。
进一步地，负载于纳米粒子或微米粒子表面的水溶性抗原和/或非水溶性抗原负载后为一层或多层，表面负载多层水溶性抗原和/或非水溶性抗原时，层与层之间为修饰物。
进一步地，用于激活抗原提呈细胞的粒子的粒径大小为纳米级或微米级，这样能保证粒子被抗原提呈细胞吞噬，而为了提高吞噬效率，粒径大小要在适宜的范围内。纳米粒子的粒径大小为1nm-1000nm，更优选地，粒径大小为30nm-1000nm，最优选地，粒径大小为50nm-600nm；微米粒子的粒径大小为1μm-1000μm，更优选地，粒径大小为1μm-100μm，更优选地，粒径大小为1μm-10μm，最优选地，粒径大小为1μm-5μm。
树突状细胞疫苗是疫苗的一种，由于DC属于活细胞制品，作为疫苗使用的被激活的DC在储存、运输和给药过程都存在诸多缺陷。本发明中，先使用负载含有自身免疫疾病抗原的细胞或组织的全细胞抗原的纳米粒子和/或微米粒子激活抗原提呈细胞，从而使抗原提呈细胞负载广谱的全细胞抗原，再将抗原提呈细胞经过机械破坏、梯度离心和/或膜过滤和/或与粒子共作用的方法处理制备成纳米疫苗或微米疫苗，所得纳米疫苗或微米疫苗负载有自身免疫疾病的抗原表位和抗原提呈细胞组分，因而易于保存、运输和使用，而且注射 进入人体后具有一定的归巢效应和易于被抗原提呈细胞吞噬、可以激活广谱多样的特异性调节性T细胞，从而可以更多和更好的抑制机体内的效应性T细胞。
借由上述方案，本发明至少具有以下优点：
(1)疫苗中可以将一种或多种抗原提呈细胞的组分与自身免疫疾病的抗原表位整合到一个纳米疫苗或微米疫苗内，一个纳米疫苗或微米疫苗内可以含有包括DC细胞、B细胞或巨噬细胞等至少一种抗原提呈细胞的组分，因而一个纳米疫苗或微米疫苗同时可以具有一种或多种抗原提呈细胞的某些功能和优势。
(2)纳米疫苗或微米疫苗中可以含有包括DC细胞在内一种或多种抗原提呈细胞的组分，注射进入体内后具有归巢淋巴结的特性，能更好的激活抗原特异性抑制性免疫反应。
(3)使用负载含有自身免疫疾病抗原的细胞和/或组织的全细胞抗原的纳米粒子和/或微米粒子激活抗原提呈细胞，再使用抗原提呈细胞制备成纳米疫苗，因而所制备的纳米疫苗可以负载用于激活抗原提呈细胞的纳米粒和/或微米粒子所负载的全部抗原表位，因为纳米疫苗可以激活广谱多样的调节性抗原特异性T细胞(Treg)。
(4)纳米疫苗或微米疫苗来源于抗原提呈细胞，组分均是生物相容性和可降解的，安全性好。
(5)将具有细胞活性的抗原提呈细胞处理后制备成无任何细胞活性的纳米疫苗或微米疫苗后，纳米疫苗或微米疫苗不像活的抗原提呈细胞那样对储存、运输和注射使用时要求条件那么苛刻。
(6)本发明在将抗原提呈细胞的膜组分包裹于递送粒子上的基础上，还将含有自身抗原的细胞的膜组分负载于纳米粒子或微米粒子表面，这样赋予了纳米疫苗或微米疫苗负载广谱抗原的能力以及表面具有细胞膜结构的能力，通过表面负载仿生膜的效应可以实现一些无仿生膜粒子无法实现的功效。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1为本发明纳米疫苗或微米疫苗的制备过程及应用示意图；其中，a为裂解含有自身免疫疾病抗原的细胞或组织后收集全细胞抗原中的水溶性组分和非水溶性组分，尔后制备纳米粒子或微米粒子的示意图；b为采用含有溶解剂的溶解液溶裂解和溶解含有自身免疫疾病抗原的细胞或组织的细胞全细胞抗原并制备纳米粒子或微米粒子的示意图；c为使用a或b中制备的上述粒子激活抗原提呈细胞后，将抗原提呈细胞制备成纳米疫苗或微米疫苗，并用该类纳米疫苗预防或治疗I型糖尿病等自身免疫疾病的示意图；
图2-15分别为实施例1-14中用纳米疫苗或微米疫苗预防或治疗I型糖尿病等自身免疫疾病时的实验结果；图3、4和14中，a为预防或治疗I型糖尿病等自身免疫疾病时的结果，b和c为使用流式细胞术分析纳米疫苗激活的CD8 +和CD4 +调节性特异性T细胞占总的CD8 +和CD4 +T细胞的比例。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好 地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明所述的用于预防或治疗I型糖尿病等自身免疫疾病的纳米疫苗或微米疫苗，其包含由被负载自身免疫疾病抗原的细胞和/或肿瘤组织全细胞抗原的纳米粒子和/或微米粒子激活的抗原提呈细胞制备所得的纳米疫苗或微米疫苗。纳米粒子和/或微米粒子负载自身免疫疾病抗原的细胞和/或组织全细胞抗原或其混合物。制备预防或治疗I型糖尿病等自身免疫疾病的纳米疫苗或微米疫苗，其制备过程及应用领域如图1所示。
在制备用于激活抗原提呈细胞的负载含有自身免疫疾病抗原的细胞和/或肿瘤组织全细胞抗原的纳米粒子或微米粒子时，可裂解细胞或组织后先分别收集水溶性抗原和水不溶性抗原并分别制备纳米或微米粒子系统；或者也可以直接采用含有溶解剂的溶解液直接裂解细胞或组织并溶解细胞全细胞抗原并制备纳米或微米粒子系统。本发明所述细胞全细胞抗原在裂解前或(和)裂解后既可经过包括但不限于灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理等处理后再制备纳米粒子或微米粒子；也可细胞裂解前或(和)裂解后不经过任何灭活或(和)变性、固化、生物矿化、离子化、化学修饰、核酸酶处理直接制备纳米粒子或微米粒子。本发明部分实施例中，组织细胞在裂解前经过了灭活或(和)变性处理，在实际使用过程中也可以在细胞裂解后做灭活或(和)变性处理，或者也可以细胞裂解前和裂解后均做灭活或(和)变性处理；本发明部分实施例中细胞裂解前或(和)裂解后的灭活或(和)变性处理方法为紫外照射和高温加热，在实际使用过程中也可以采用包括但不限于放射线辐照、高压、固化、生物矿化、离子化、化学修饰、核酸酶处理、胶原酶处理、冷冻干燥等处理方法。本领域技术人员可以理解，在实际应用过程中技术人员可根据具体情况进行适当调整。
在使用纳米粒子或微米粒子体外激活抗原提呈细胞时，可以使用细胞因子和/或抗体辅助提高激活效率，抗原提呈细胞可以来源于自体或者同种异体，也可以来自于细胞系或者干细胞。抗原提呈细胞可以是DC细胞、B细胞、巨噬细胞或者上述三者的任意混合物，也可以是其他具有抗原提呈功能的细胞。
在抗原提呈细胞被激活后，使用机械破坏细胞后，采用离心和/或膜过滤和/或与纳米粒子或微米粒子共作用等方法将抗原提呈细胞制备成纳米疫苗或微米疫苗。在实际制备是也可以采用其他可以将活的抗原提呈细胞制备成不具有细胞活性的纳米疫苗或微米疫苗的方法。
在一些实施方案中，采用负载含有自身免疫疾病抗原的细胞和/或组织全细胞抗原的纳米粒子或微米粒子激活的抗原提呈细胞后将抗原提呈细胞制备成纳米疫苗或微米疫苗的具体制备方法如下：
步骤1，将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度制备粒子原材料的有机相中。
在一些实施例中，水相溶液可含有细胞裂解物中的各组分以及免疫抑制剂；细胞裂解物中的各组分在制备时分别为水溶性抗原或是溶于尿素或盐酸胍等溶解剂中的原非水溶性抗原。水相溶液所含有的水溶性抗原的浓度或原非水溶性抗原的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL，能负载足够细胞全细胞抗原以激活相关细胞。免疫抑制剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中，水相溶液含有组织裂解物中的各组分以及免疫抑制剂；组织裂解物 中的各组分在制备时分别为水溶性抗原或者是溶于尿素或盐酸胍等溶解剂中的原非水溶性抗原。水相溶液所含有的水溶性抗原的浓度或原非水溶性抗原的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于0.01ng/mL，能负载足够全细胞抗原以激活相关细胞。免疫抑制剂在初始水相中的浓度为大于0.01ng/mL。
在一些实施例中，制备粒子原材料为聚乳酸-羟基乙酸共聚物(PLGA)或聚乳酸(PLA)，有机溶剂选用二氯甲烷。另外，在一些实施例中，制备粒子原材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL，优选为100mg/mL。
在本发明中，之所以选择PLGA、PLA或修饰的PLGA、PLA，是由于该材料为生物可降解材料且已被FDA批准用作药物辅料。研究表明PLGA和PLA具有一定的免疫调节功能，因而适合作为纳米粒子或微米粒子制备时的辅料。在实际应用中可根据实际情况选择合适的材料。
实际中，有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定，在本发明中，水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000，优选地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒或微米粒的尺寸大小。
优选地，水相溶液为裂解物组分溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL；水相溶液为裂解物组分/免疫抑制剂溶液时，其中蛋白质和多肽的浓度大于1ng/mL，优选1mg/mL～100mg/mL，免疫佐剂的浓度大于0.01ng/mL，优选0.01mg/mL～20mg/mL。有机相溶液中，溶剂为DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等，优选二氯甲烷；有机相的浓度为0.5mg/mL～5000mg/mL，优选为100mg/mL。
步骤2，将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌或均质处理或微流控处理。优选地，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于1分钟，比如搅拌速度为50rpm～1500rpm，搅拌时间为0.1小时～24小时；超声处理时，超声功率大于5W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于5psi，比如20psi～100psi，使用高剪切均质机时转速大于100rpm，比如1000rpm～5000rpm；使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化和/或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的微纳粒子大小，过大或过小都会带来粒径大小的变化。
步骤3，将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌或进行均质处理或微流控处理。该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化或微米化。在本发明中，超声时间大于0.1秒，比如2～200秒，搅拌速度大于50rpm，比如50rpm～500rpm，搅拌时间大于1分钟，比如60～6000秒。优选地，搅拌为机械搅拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于1分钟，比如搅拌速度为50rpm～1500rpm，搅拌时间为0.5小时～5小时；超声处理时，超声功率为50W～500W，时间大于0.1秒，比如2～200秒；均质处理时使用高压/超高压均质机或高剪切均质机，使用高压/超高压均质机时压力大于20psi，比如20psi～100psi，使用高剪切均质机时转速大于1000rpm，比如1000rpm～ 5000rpm；使用微流控处理流速大于0.01mL/min,比如0.1mL/min-100mL/min。超声或者搅拌或者均质处理或者微流控处理进行纳米化或微米化，超声时间长短或搅拌速度或均质处理压力及时间能控制制备的纳米或微米粒子大小，过大或过小都会带来粒径大小的变化。
在一些实施例中，乳化剂水溶液为聚(乙烯-alt-马来酸酐)(PEMA)水溶液或者聚乙烯醇(PVA)，第三预定体积为5mL，第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中，第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定，优选地可以为2:5。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。同样地，本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒或微米粒。
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。
本步骤中，乳化剂水溶液为PEMA或PVA溶液或其他溶液。
第四预定浓度为5mg/mL，第四预定浓度的选择，以得到尺寸大小合适的纳米粒或微米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中，第三预定体积与第三预定体积之比为范围为1:1.5-1:2000，优选地为1:10。在具体实施过程中为了控制纳米粒子或微米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中，本步骤的预定搅拌条件为直至有机溶剂挥发完成，也即步骤1中的二氯甲烷挥发完成。
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
在本发明一些实施方案中，步骤5所得沉淀重新混悬于第六预定体积的PBS(或生理盐水)中时不需要冻干，可直接进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验。
在本发明一些实施方案中，步骤5所得沉淀重新混悬于含有冻干保护剂的水溶液中时需进行冷冻干燥，再冷冻干燥以后再进行后续纳米粒子或微米粒子表面吸附癌细胞裂解物的相关实验。
在本发明中，所述冻干保护剂选用海藻糖(Trehalose)。
在本发明中，该步骤的冻干保护剂的第五预定浓度为质量百分比4％，之所以如此设定，是为了在后续进行冷冻干燥中不影响冻干效果。
步骤6，将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后，将冻干物质备用。
步骤7，将第六预定体积的步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第六预定体积的PBS(或生理盐水)重悬步骤6得到的冷冻干燥后的含有纳米粒或微米粒和冻干保护剂的冻干物质直接使用；或者上述样品与第七预定体积的水溶性抗原或者溶解的原非水溶性抗原混合后使用。
在本发明中，第六预定体积与第七预定体积的体积比为1:10000到10000:1，优先体积 比为1:100到100:1，最优体积比为1:30到30:1。
在一些实施例中，所述重悬的纳米粒子混悬液体积为10mL时，含有细胞裂解物或含有组织裂解物中的水溶性抗原或者溶解的原非水溶性抗原的体积与为1mL。在实际使用时可将二者体积和比例根据需要进行调整。
步骤8，将抗原提呈细胞与上述制备的纳米粒子和/或微米粒子共孵育一定时间。制备纳米粒子和/微米粒子的组织和/或细胞与抗原提呈细胞可以来自于自体或者同种异体。
步骤9，收集共孵育后的抗原提呈细胞和/或培养的含有自身抗原的细胞，比如β细胞，可以进行洗涤或不经洗涤然后超声、机械搅拌、均质化等处理。
步骤10，将机械处理后的抗原提呈细胞和/或含有自身抗原的细胞样品进行梯度离心、和/或膜过滤、和或与纳米粒子或微米粒子共作用等处理，制备得到纳米疫苗或微米疫苗。
在另一些实施方案中，制备纳米疫苗或微米疫苗的方法如下：
步骤1～4同上。
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有细胞全细胞抗原中水溶性和/或非水溶性抗原的溶液中，或者将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有细胞全细胞抗原中水溶性和/或非水溶性抗原与免疫抑制剂混合的溶液中。
步骤6，将步骤5处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第六预定体积的固化处理试剂或矿化处理试剂,作用一定时间后离心洗涤，然后加入第七预定提交的含有带正电或者带负电的物质并作用一定时间。
在本发明一些实施方案中，步骤6所得沉淀重新混悬于第七预定体积的带电物质后可不需要冻干，可直接进行后续纳米粒子或微米粒子表面负载细胞/组织裂解物的相关实验。
在本发明一些实施方案中，步骤6所得沉淀重新混悬于含有干燥保护剂的水溶液中后进行室温真空干燥或者冷冻真空干燥，在干燥以后再进行后续纳米粒子或微米粒子表面吸附细胞裂解物的相关实验。
在本发明中，所述冻干保护剂选用海藻糖(Trehalose)，或者甘露醇与蔗糖的混合溶液。在本发明中，该步骤的干燥保护剂的浓度为质量百分比4％，之所以如此设定，是为了在后续进行干燥中不影响干燥效果。
步骤7，将步骤6得到的含有干燥保护剂的混悬液进行干燥处理后，将干燥后的物质备用。
步骤8，将第八预定体积的步骤6中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第八预定体积的PBS(或生理盐水)重悬步骤7得到的干燥后的含有纳米粒或微米粒和干燥保护剂的干燥后物质直接使用；或者与第九预定体积的水溶性抗原或者非水溶性抗原混合后使用。
在本发明中，步骤5-步骤8的修饰和抗原负载步骤可重复多次以提高抗原的负载量。而且在添加带正电或带负电的物质时可以多次添加带同种电荷的或者也可以交替添加带不同电荷的物质。
在一些实施例中，所述重悬的纳米粒子混悬液体积为10mL时，含有细胞裂解物或含有组织裂解物中的水溶性抗原或者原非水溶性抗原的体积与为0.1-100mL。在实际使用时可将二者体积和比例根据需要进行调整。
步骤9，将抗原提呈细胞与上述制备的纳米粒子和/或微米粒子共孵育一定时间。制备纳米粒子和/微米粒子的组织和/或细胞与抗原提呈细胞可以来自于自体或者同种异体。
步骤10，收集共孵育后的抗原提呈细胞和/或培养的含有自身抗原的细胞，比如β细胞，可以进行洗涤或不经洗涤然后超声、机械搅拌、均质化等处理。
步骤11，将机械处理后的抗原提呈细胞和/或含有自身抗原的细胞样品进行梯度离心、和/或膜过滤、和或与纳米粒子或微米粒子共作用等处理，制备得到纳米疫苗或微米疫苗。
实施例1基于DC细胞细胞膜的纳米疫苗用于I型糖尿病的预防
NIT-1细胞为小鼠β细胞模型，可以用来作为胰岛β细胞使用。首先裂解NIT-1细胞制备NIT-1细胞的水溶性组分和非水溶性组分。然后，以有机高分子材料PLA为纳米粒骨架材料，以雷帕霉素为免疫抑制剂采用溶剂挥发法制备负载有β细胞的水溶性组分和非水溶性组分的纳米粒子，然后应用该粒子激活树突状细胞(DC)，使树突状细胞向着激活调节性T细胞的方法激活，然后将树突状细胞超声处理后制备成纳米疫苗预防I型糖尿病。
(1)β细胞的裂解和各组分的收集
在高糖培养基中培养NIT-1β细胞后，收集所培养的NIT-1β细胞，离心后去除培养基，用超纯水重悬NIT-1β细胞，然后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融3次以上，伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以3000g离心5分钟，并取上清液即为NIT-1β细胞可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素(含500mM氯化钠)溶解沉淀部分即可将NIT-1β细胞中不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。上述所得来源于细胞裂解物的水溶性组分和溶解于8M尿素中的原非水溶性组分即为制备激活DC的纳米粒子的抗原。
(2)纳米粒子的制备
本实施例中制备纳米粒采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料聚乳酸(PLA)分子量为30KDa，所采用的免疫抑制剂为雷帕霉素且雷帕霉素制备负载于纳米粒子内部，在制备时雷帕霉素与PLGA一同溶解于有机相中，所使用的乳化剂为PEMA。制备方法如前所述。水溶性组分和非水溶性组分分别负载于不同的纳米粒子，然后使用时混合后一起使用。在制备过程中首先采用复乳法在纳米粒子内部负载全细胞裂解物组分和免疫抑制剂，在内部负载细胞裂解组分和佐剂后，将100mg纳米粒子在10000g离心20分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h，在使用前将20mg冻干的纳米粒子重悬于0.9mLPBS中，然后与0.1mg裂解物组分(60mg/mL)混合后在室温作用3分钟后即得内外都负载全细胞抗原的纳米粒子。纳米粒子平均粒径为300nm，表面电位为-3mV左右。每1mgPLA纳米粒子约负载130μg蛋白质或多肽组分，每1mgPLA纳米粒子负载雷帕霉素约为0.05mg。空白纳米粒子制备材料和制备方法相同，粒径为300nm,负载等量免疫抑制剂但是不负载任何裂解物组分。
(3)骨髓来源的树突状细胞(BMDC)的制备
本实施例以从小鼠骨髓细胞制备树突状细胞为例来说明如何制备BMDC。首先，取1 只6-8周龄C57小鼠颈椎脱臼处死，手术取出后腿的胫骨和股骨放入PBS中，用剪刀和镊子将骨周围的肌肉组织剔除干净。用剪刀剪去骨头两端，再用注射器抽取PBS溶液，针头分别从骨头两端插入骨髓腔，反复冲洗骨髓到培养皿中。收集骨髓溶液，400g离心3min后加入1mL红细胞裂解液裂红。加入3mLRPMI 1640(10％FBS)培养基终止裂解，400g离心3min，弃上清。将细胞放置10mm培养皿中培养，使用RPMI 1640(10％FBS)培养基，同时加入重组小鼠GM-CSF(20ng/mL)，37度，5％CO 2培养7天。第3天轻轻摇晃培养瓶，补充同样体积含有GM-CSF(20ng/mL)RPMI 1640(10％FBS)培养基。第6天，对培养基进行半量换液处理。第7天，收集少量悬浮及半贴壁细胞，通过流式检测，当CD86 +CD80 +细胞在CD11c +细胞中的比例为15-20％之间，诱导培养的BMDC即可被用来做下一步实验。
(4)DC的激活
将负载来源于β细胞全细胞抗原的纳米粒子(500μg，其中负载水溶性组分的纳米粒子250μg，负载非水溶性组分的纳米粒子250μg)或空白纳米粒(500μg)+游离裂解液与BMDC(1000万个)在15mLRPMI1640完全培养基中共孵育96小时(37℃，5％CO 2)；孵育体系中含有细胞因子组合：粒细胞－巨噬细胞集落刺激因子(GM-CSF，2000U/mL)、IL-2(200U/mL)、IL-4(500U/mL)、IL-7(200U/mL)、IL-10(1000U/mL)。
(5)DC来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的DC，然后使用PBS洗涤细胞两遍，将细胞重悬在PBS中后在7.5W超声20分钟。然后将样品在2000g离心20分钟并收集上清液，将上清液在7000g离心20分钟后收集上清液后使用0.22μm的滤膜过滤挤出后收集滤液，然后将滤液在15000g离心120分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得基于DC细胞细胞膜的纳米疫苗，纳米疫苗粒径为120纳米。使用负载全细胞抗原的纳米粒子激活的DC制备的纳米疫苗为纳米疫苗1；使用空白纳米粒+游离裂解液激活的DC制备的纳米疫苗为纳米疫苗2。
或者将步骤(4)激活的DC收集后在400g离心5分钟，然后使用PBS洗涤细胞两遍，将细胞重悬在PBS后直接作为DC疫苗3使用。
(6)纳米疫苗或DC疫苗预防小鼠I型糖尿病
本研究对照组是PBS组和空白纳米粒+细胞裂解物组。选取3周龄雌性NOD小鼠进行本实验。在实验中，每组10只NOD小鼠,从第三周开始每隔7天皮下注射100μg纳米疫苗1、100μg纳米疫苗2或者500万个DC疫苗3，连续给6周。PBS对照组从第三周开始每隔7天皮下注射100μLPBS，连续给6周。各组小鼠从第8周开始每天记录小鼠血糖情况。以血糖高于11.0mmol·L-1为糖尿病开始发病。记录不同时间段NOD小鼠糖尿病发病情况。
(7)实验结果
NOD小鼠为I型糖尿病模型小鼠。有大约70％-85％的雌性NOD小鼠在未经预防处理的情况下会在24周后罹患I型糖尿病。如图2所示，PBS或空白纳米粒+细胞裂解物激活的抗原提呈细胞制备的纳米疫苗2处理的小鼠在25周后有70％-80％患有糖尿病；使用负载全细胞抗原的活细胞DC疫苗处理的小鼠有大概50％患有I型糖尿病；使用负载β细胞全 细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗1处理的小鼠，在25周后只有约40％小鼠会患有I型糖尿病。这说明，本发明所述的被负载β细胞全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗对I型糖尿病具有良好预防效果，且预防效果优于纳米粒子激活的抗原提呈细胞活疫苗。
实施例2抗原提呈细胞制备纳米疫苗用于I型糖尿病的预防
(1)β细胞的裂解和各组分的收集
在高糖培养基中培养NIT-1β细胞后，收集所培养的NIT-1β细胞，离心后去除培养基，用超纯水重悬NIT-1β细胞，然后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融3次以上，伴有超声以破坏裂解细胞。待细胞裂解完成后，将裂解物以2000g离心10分钟，并取上清液即为NIT-1β细胞可溶于纯水的水溶性组分；在所得沉淀部分中加入10％脱氧胆酸钠溶解沉淀部分即可将NIT-1β细胞中不溶于纯水的非水溶性组分转化为在水溶液中可溶。上述所得来源于细胞裂解物的水溶性组分和溶解于脱氧胆酸钠中的原非水溶性组分按质量比1:1混合后，即为制备激活抗原提呈细胞的粒子的抗原组分。
(2)微米粒子系统的制备
本实施例中制备微米粒子采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫抑制剂为雷帕霉素和他克莫司混合免疫抑制剂，且混合免疫抑制剂分布于微米粒子内部，在制备时雷帕霉素、他克莫司与PLGA一同溶解于有机相中。制备方法如前所述，在制备过程中首先采用复乳法在微米粒子内部负载β细胞全细胞裂解物组分和免疫抑制剂，在内部负载细胞裂解组分和免疫抑制剂后，将100mg微米粒子在7000g离心10分钟，弃去上清液后将沉淀使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h备用。所得微米粒子平均粒径为1.5μm左右，微米疫苗表面电位为-7mV左右，每1mgPLGA微米疫苗约负载90μg蛋白质或多肽组分，每1mgPLGA微米疫苗所负载的雷帕霉素和他克莫司各为0.025mg。空白微米粒子粒径为1.4μm左右，空白微米粒制备材料和制备方法相同，负载等量雷帕霉素和他克莫司但是不负载任何裂解后的水溶性组分和非水溶性组分。负载几种I型糖尿病抗原多肽的微米粒制备方法相同，所负载的质量等量的多肽为Insulin B 9-23,InsulinA 14-20,IGRP 206-214,和GAD 225-244，粒子平均粒径为1.5μm左右，微米疫苗表面电位为-7mV左右，每1mg PLGA微米粒子约负载90μg多肽组分和等量的免疫抑制剂。
(3)抗原提呈细胞的制备
本实施例以从小鼠骨髓细胞制备树突状细胞为例来说明如何制备BMDC。首先，取1只6-8周龄C57小鼠颈椎脱臼处死，手术取出后腿的胫骨和股骨放入PBS中，用剪刀和镊子将骨周围的肌肉组织剔除干净。用剪刀剪去骨头两端，再用注射器抽取PBS溶液，针头分别从骨头两端插入骨髓腔，反复冲洗骨髓到培养皿中。收集骨髓溶液，400g离心3min后加入1mL红细胞裂解液裂红。加入3mL RPMI 1640(10％FBS)培养基终止裂解，400g离心3min，弃上清。将细胞放置10mm培养皿中培养，使用RPMI 1640(10％FBS)培养基，同时加入重组小鼠GM-CSF(20ng/mL)，37度，5％CO 2培养7天。第3天轻轻摇晃培养瓶，补充同样体积含有GM-CSF(20ng/mL)RPMI 1640(10％FBS)培养基。第6天，对培养基进行半量换液处理。第7天，收集少量悬浮及半贴壁细胞，通过流式检测，当CD86 +CD80 + 细胞在CD11c +细胞中的比例为15-20％之间，诱导培养的BMDC即可被用来做下一步实验。
处死NOD小鼠后摘取小鼠脾脏，制备小鼠脾细胞单细胞悬液，使用磁珠分选法分离脾细胞活细胞中(使用活死细胞染料标记死细胞以去除死细胞)的CD19 +B细胞备用。
(4)抗原提呈细胞的激活
将负载全细胞抗原的微米粒子(500μg)或多肽微米粒子(500μg)或空白微米粒(500μg)+游离裂解液与BMDC(500万个)和B细胞(500万个)在15mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO 2)，孵育体系中含有GM-CSF(2000U/mL)、IL-2(200U/mL)、IL-7(1000U/mL)、IL-10(2000U/mL)和CD40抗体(20ng/mL)。
(5)基于抗原提呈细胞的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的抗原提呈细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声3分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，然后在18000g离心60分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后使用0.22μm膜过滤器过滤样品即得纳米疫苗，纳米疫苗粒径为120纳米。
(6)纳米疫苗预防小鼠I型糖尿病
同实施例1。
(7)抗原特异性调节性T细胞(T reg)的分析
选取6-8周的雌性C57BL/6为模型小鼠,在第0天、第7天、第14天、第28天和第42天分别每只小鼠皮下注射100μg纳米疫苗或PBS。在第45天处死小鼠，摘取小鼠脾脏并制备脾细胞单细胞悬液，使用磁珠分选法从小鼠脾细胞中分选出B细胞和T细胞。将100μg负载β细胞全细胞抗原的微米粒子、500万个B细胞和100万个T细胞在5mL RPMI1640完全培养基中共孵育48小时(37℃,5％CO 2)。然后收集孵育后细胞并标记活死细胞染料、CD3抗体、CD8抗体、CD4抗体、CD25抗体、Ly49抗体和FOXP3抗体，尔后流式细胞术分析T细胞亚群中CD4 +CD25 +FOXP3 +T细胞占CD4 +T细胞的比例和CD8+Ly49 +T占CD8 +T细胞的比例。微米粒子所负载的β细胞全细胞抗原在被抗原提呈细胞B细胞吞噬后可被降解成抗原表位被提呈到抗原提呈细胞表面，可以识别β细胞全细胞抗原的特异性T细胞即可以识别β细胞全细胞抗原表位后被激活并高表达特异性表面标志物。使用流式细胞术分析的高表达特定标志物的T细胞即为调节性T细胞，亦即可以抑制识别和杀伤β细胞的效应性T细胞(T eff)的T reg。
(8)实验结果
如图3a所示，PBS或者空白微米粒+细胞裂解物激活的抗原提呈细胞制备的纳米疫苗处理的小鼠在25周后有70％-80％患有I型糖尿病；负载抗原多肽的微米粒激活的抗原提呈细胞制备的纳米疫苗处理的小鼠在25周后有50％患有糖尿病。在采用负载β细胞全细胞抗原的微米粒子激活的抗原提呈细胞制备的微米疫苗处理的小鼠在25周后只有约30％小鼠患有I型糖尿病。综上所述，本发明所述的纳米疫苗对I型糖尿病具有预防效果。这说明负载抗原多肽微米粒子激活的DC+B细胞所提呈的抗原表位种类有限，因而其制备的纳米疫苗所能激活的β细胞特异性T reg细胞系统所含有的T细胞克隆数很少，所能抑制的与T reg识别相同抗原的特异性T eff也就种类较少。而负载β细胞全细胞抗原的微米粒子激活的DC+B细胞提呈的β细胞抗原更广谱，因而其制备的纳米疫苗所能激活的T reg细胞克隆数也 就更广谱，所能抑制的与T reg识别相同抗原的特异性T eff种类也很广谱，预防I型糖尿病的效果也越好。
如图3b，负载全细胞抗原的微米粒子激活的DC+B细胞制备的纳米疫苗所能诱导的CD8 +Ly49 +T细胞和CD4 +CD25 +FOXP3 +T细胞分别占CD8 +T细胞和CD4 +T细胞的比例明显高于负载多肽的微米粒子和空白微米粒子+游离裂解液激活的DC+B细胞制备的纳米疫苗所诱导的比例。由此可见，本发明所述的负载全细胞抗原的微米粒子激活的抗原提呈细胞制备的纳米疫苗可以更好的激活具有识别和抑制T eff的β细胞特异性T reg细胞。
实施例3抗原提呈细胞来源的微米疫苗用于I型糖尿病的预防
本实施例以小鼠胰岛组织裂解物全细胞组分和β细胞裂解物全细胞组分混合物制备微米粒子，然后以该微米粒子激活抗原提呈细胞，再使用抗原提呈细胞制备微米疫苗。
(1)胰岛和β细胞的裂解和各组分的收集
处死小鼠后收集小鼠胰岛组织，将胰岛组织通过细胞过滤网后制备成单细胞悬液，并加入适量纯水后反复冻融5次，并伴有超声以破坏裂解细胞，将裂解物以3000g的转速离心3分钟并取上清液即为水溶性组分，将沉淀部分使用8M尿素水溶液(含500mM氯化钠)溶解即为溶解剂溶解够的非水溶性组分。
在高糖DMEM完全培养基(葡萄糖含量为正常含量的4倍)中培养NIT-1细胞24小时，并在培养过程中加入10μg毒胡萝卜素，然后收集所培养的NIT-1细胞，离心后去除培养基，用超纯水重悬NIT-1细胞，然后加一定量超纯水后反复冻融5次，伴有超声以破坏裂解细胞。待细胞裂解完成后，将裂解物以2000g离心10分钟，并取上清液即为NIT-1细胞可溶于纯水的水溶性组分。将沉淀部分使用8M尿素水溶液(含500mM氯化钠)溶解即为溶解剂溶解后的非水溶性组分。高葡萄糖条件和使用毒胡萝卜素均可以增加NIT-1细胞合成各类抗原和胰岛素颗粒的量，从而增加细胞内所含邮的各类抗原含量。
上述来源于β细胞裂解物的水溶性组分和来源于胰岛组织裂解物的水溶性组分按质量比1:1混合后即为水溶性组分混合物；来源于β细胞裂解物的非水溶性组分和来源于胰岛组织裂解物的非水溶性组分按质量比1:1混合后即为非水溶性组分混合物；将水溶性组分混合物和非水溶性组分混合物按质量比2:1混合物，即为全细胞组分混合物，含有I型糖尿病相关的全细胞抗原。
(2)微米粒子系统的制备
本实施例中制备微米粒子采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料PLA分子量为40KDa，所采用的免疫抑制剂为环孢素A和他克莫司混合免疫抑制剂，所使用的增加溶酶体逃逸物质为NH 4HCO 3，而且混合免疫抑制剂和增加溶酶体逃逸的物质都分布于微米粒子内部，在制备时环孢素A、他克莫司与PLA一同溶解于有机相中，NH 4HCO 3与裂解物组分一起溶解于第一水相。制备方法如前所述，在制备过程中首先采用复乳法在微米粒子内部负载全细胞裂解物组分、NH 4HCO 3和免疫抑制剂，然后将100mg微米粒子在6000g离心10分钟，弃去上清液后将沉淀使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h备用；在使用前将其用9mL PBS重悬然后加入1mL的裂解液组分(蛋白质浓度80mg/mL)并室温作用10min，得到内外都负载全细胞裂解物组分的微米粒子系统。所得微米粒子平均粒径为2.55μm左右，微米疫苗表面电位为-4mV左右，每1mg PLA微米疫苗约负载130μg蛋白质或多肽组分，每1mgPLA微米疫苗负载的环孢素A和他克莫 司各0.025mg，负载NH 4HCO 3为0.05mg。
(3)抗原提呈细胞的制备
本实施例使用来源于外周血的DC细胞以及B细胞作为抗原提呈细胞。处死小鼠后收集小鼠外周血，利用梯度离心法分离小鼠外周血单核细胞(PBMC)，然后使用流式细胞术从小鼠PBMC中分选出CD19 +B细胞和CD11c +DC,将上述B细胞和DC按数量比1:1混合即为所用混合抗原提呈细胞。
(4)抗原提呈细胞的激活
将负载全细胞抗原的微米粒子(500μg)与混合抗原提呈细胞(共1000万个细胞，其中含500万个DC和500万个B细胞)在15mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO2)，孵育体系中含有细胞因子组合1:TGF-β(2000U/mL)、IL-4(200U/mL)、IL-7(1000U/mL)、IL-10(2000U/mL)。
或者将负载全细胞抗原的微米粒子(500μg)与混合抗原提呈细胞(共1000万个细胞，其中含500万个DC和500万个B细胞)在15mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO2)，孵育体系中含有细胞因子组合2:TNF-a(2000U/mL)、IL-12(200U/mL)、IL-6(1000U/mL)、IL-15(2000U/mL)。
或者将混合抗原提呈细胞(共1000万个细胞，其中含500万个DC和500万个B细胞)在15mL高糖DMEM完全培养基中孵育72小时(37℃，5％CO2)，孵育体系中含有细胞因子组合1:TGF-β(2000U/mL)、IL-4(200U/mL)、IL-7(1000U/mL)、IL-10(2000U/mL)。
(5)基于抗原提呈细胞或β细胞细胞膜的微米疫苗的制备
通过在300g离心4分钟收集步骤(4)激活后的混合抗原提呈细胞(1000万个，其中含500万个DC和500万个B细胞)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在低功率(5W)超声30分钟。然后将样品在依次过孔径为10μm、5μm、2μm、1μm和0.45μm的膜过滤后收集滤液，将滤液在16000g离心20分钟后弃去沉淀收集上清液，将上清液与滤液与步骤(2)制备的微米粒子(80mg)混合后在20W超声处理3分钟后共孵育15分钟后使用5μm的滤膜过滤挤出后收集滤液，然后将滤液在8000g离心15分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得微米疫苗。其中，使用微米粒子与混合抗原提呈细胞共孵育过程中加入细胞因子组合1所激活的混合抗原提呈细胞所制备的微米疫苗为微米疫苗1，粒径为2.60μm；使用微米粒子与混合抗原提呈细胞共孵育过程中加入细胞因子组合2所激活的混合抗原提呈细胞所制备的微米疫苗为微米疫苗2，粒径为2.60μm；仅将混合抗原提呈细胞与细胞因子组合1共孵育而不加入任何微米粒子的混合抗原提呈细胞所制备的微米疫苗为微米疫苗3，粒径为2.60μm。
或者收集所培养的NIT-1细胞(1000万个)，离心后去除培养基，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在低功率(5W)超声30分钟。然后将样品在依次过孔径为10μm、5μm、2μm、1μm和0.45μm的膜过滤后收集滤液，将滤液在16000g离心20分钟后弃去沉淀收集上清液，将上清液与步骤(2)制备的微米粒子(80mg)混合后在20W超声处理3分钟后共孵育15分钟后使用5μm的滤膜过滤挤出后收集滤液，然后将滤液在8000g离心15分钟后弃去上清液 收集沉淀，将沉淀在PBS中重悬后即得微米疫苗4，粒径为2.60μm。
或者同时收集步骤(4)激活后的混合抗原提呈细胞(500万个，其中250万个DC细胞+250万个B细胞)和NIT-1细胞(500万个)并混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在低功率(5W)超声30分钟。然后将样品在依次过孔径为10μm、5μm、2μm和1μm的膜过滤后收集滤液，将滤液在16000g离心20分钟后弃去沉淀收集上清液，将上清液与步骤(2)制备的微米粒子(80mg)混合后在20W超声处理3分钟后共孵育15分钟后使用5μm的滤膜过滤挤出后收集滤液，然后将滤液在8000g离心15分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得微米疫苗5，粒径为2.60μm。
(6)微米疫苗预防小鼠I型糖尿病
本研究对照组是PBS组。选取3周龄雌性NOD小鼠进行本实验。在实验中，每组10只NOD小鼠,从第三周开始每隔7天皮下注射100μg微米疫苗1、或者100μg微米疫苗2、或者100μg微米疫苗3，或者100μg微米疫苗4，或者100μg微米疫苗5，或者100μL PBS，连续给6周。各组小鼠从第8周开始每天记录小鼠血糖情况。以血糖高于11.0mmol·L -1为糖尿病开始发病。记录不同时间段NOD小鼠糖尿病发病情况。
(7)抗原特异性调节性T细胞(T reg)的分析
选取6-8周的雌性C57BL/6为模型小鼠,在第0天、第7天、第14天、第28天和第42天分别每只小鼠皮下注射100μg微米疫苗1、或者微米疫苗2、或者微米疫苗3、或者微米疫苗4或者微米疫苗5。在第45天处死小鼠，摘取小鼠脾脏并制备脾细胞单细胞悬液，使用磁珠分选法从小鼠脾细胞中分选出B细胞和T细胞。将100μg负载β细胞全细胞抗原的微米粒子、500万个B细胞和100万个T细胞在5mL RPMI1640完全培养基中共孵育48小时(37℃,5％CO 2)。然后收集孵育后细胞并标记活死细胞染料、CD3抗体、CD8抗体、CD4抗体、CD25抗体、Ly49抗体和FOXP3抗体，尔后流式细胞术分析T细胞亚群中CD4 +CD25 +FOXP3 +T细胞占CD4 +T细胞的比例和CD8+Ly49 +T占CD8 +T细胞的比例。微米粒子所负载的β细胞全细胞抗原在被抗原提呈细胞B细胞吞噬后可被降解成抗原表位被提呈到抗原提呈细胞表面，可以识别β细胞全细胞抗原的特异性T细胞即可以识别β细胞全细胞抗原表位后被激活并高表达特异性表面标志物。使用流式细胞术分析的高表达特定标志物的T细胞即为调节性T细胞，亦即可以抑制识别和杀伤β细胞的效应性T细胞(T eff)的T reg。
(8)实验结果
如图4a所示，PBS对照组小鼠在25周后有80％患有I型糖尿病。各个微米疫苗处理的小鼠患有I型糖尿病的比例都明显降低。说明本实施例制备的各类型微米疫苗都对I型糖尿病具有良好的预防效果。其中微米疫苗2处理组小鼠在25周后有40％患有糖尿病；微米疫苗3处理组小鼠在25周后有约30％小鼠患有I型糖尿病；微米疫苗1处理组小鼠在25周后有10％小鼠患I型糖尿病；微米疫苗4处理组小鼠在25周后有20％小鼠患I型糖尿病；微米疫苗5处理组小鼠在25周后没有小鼠患I型糖尿病。这说明粒子表面负载有被负载全细胞抗原的粒子激活的混合抗原提呈细胞细胞膜组分，可以明显提高疫苗预防I型糖尿病的功效；而且，粒子表面负载有含有I型糖尿病相关抗原的细胞的细胞膜组分也可以提高微米疫苗预防I型糖尿病的功效；而且，当粒子表面同时负载混合抗原提呈细胞细胞膜组分和含有抗原的细胞膜组分时预防I型糖尿病的效果最佳。微米疫苗1明显优于微 米疫苗3说明在将抗原提呈细胞细胞膜组分负载在粒子表面前将抗原提呈细胞使用负载细胞全细胞抗原的粒子激活可以明显提高最后所制备的粒子的疗效。而且，微米疫苗1优于微米疫苗2说明在使用粒子激活混合抗原提呈细胞时加入特定细胞因子有助于提高抗原提呈细胞细胞膜所制备的疫苗的功效。综上所述，本发明所述的微米疫苗对I型糖尿病具有预防效果。
如图4b和4c所示，微米疫苗1所能诱导的自身抗原特异性T reg多于微米疫苗2和微米疫苗3；而微米疫苗5诱导的自身抗原特异性T reg多于微米疫苗1。这说明内部负载细胞全细胞抗原抗原、表面负载被激活抗原提呈细胞的细胞膜尤其是混合抗原提呈细胞的细胞膜都有利于诱导产生更多的自身抗原特异性T reg。因为可以抑制具有细胞杀伤能力的T eff，所以所诱导的就能对预防或治疗I型糖尿病有疗效。
本实施例使用的是微米疫苗，纳米疫苗也适用。本实施例以自身免疫疾病中的I型糖尿病为例，在实践中，其他自身免疫疾病也适用。
本实施例使用了高葡萄糖条件和使用毒胡萝卜素增加含有与自身免疫疾病抗原的细胞合成各类抗原的量，在实际应用时也可以使用任何其他增加细胞内抗原合成量的方法，比如增加细胞内钙离子含量等方法。
实施例4纳米疫苗用于I型糖尿病的预防
本实施例以小鼠I型糖尿病模型来说明如何使用抗原提呈细胞来源的纳米疫苗预防自身免疫疾病。本实施例中，首先裂解胰腺组织以制备胰腺组织的水溶性抗原和非水溶性抗原；然后，制备负载胰腺组织的水溶性抗原和非水溶性抗原的纳米粒子系统。在本实施例中采用了硅化和添加带电物质的方法来增加抗原的负载量，且只进行了一轮矿化处理。本实施例中，先使用纳米粒子激活抗原提呈细胞，然后使用抗原提呈细胞制备纳米疫苗。
(1)胰腺组织的裂解及各组分的收集
处死小鼠后摘取小鼠胰腺组织。将胰腺组织切块后研磨，加入胶原酶在RPMI 1640培养基中孵育30min,然后通过细胞过滤网制备单细胞悬液，并加入适量纯水并反复冻融5次，伴有超声以破坏裂解所得细胞。待细胞裂解后，将裂解物以5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性抗原；在所得沉淀部分中加入10％的十二烷基硫酸钠(SDS)水溶液溶解沉淀部分即可将不溶于纯水的非水溶性抗原转化为在10％SDS水溶液中可溶，将水溶性抗原和非水溶性抗原按质量比2:1混合，即为制备粒子的抗原原料来源。
(2)纳米粒子的制备
本实施例中纳米粒子及作为对照的空白纳米粒采用溶剂挥发法制备，并进行了适当的修饰改进，在纳米粒子制备过程中采用低温硅化技术和添加带电物质两种修饰方法提高抗原的负载量。所采用的纳米粒子制备材料PLA分子量为40KDa，所采用的免疫抑制剂为芬戈莫德，芬戈莫德与PLA溶解于有机相中。制备方法如前所述，在制备过程中首先采用复乳法在纳米粒子内部负载抗原和免疫抑制剂，在内部负载裂解组分后，将100mg纳米粒子在10000g离心20分钟，然后使用7mL PBS重悬纳米粒子并与3mL含有细胞裂解物(60mg/mL)的PBS溶液混合，尔后在10000g离心20分钟，然后采用10mL硅酸盐溶液(含150mM NaCl、80mM原硅酸四甲酯和1.0mM HCl，pH 3.0)重悬,并在室温固定10min，尔后在-80℃固定24h，使用超纯水离心洗涤后使用3mL含鱼精蛋白(5mg/mL)和聚赖氨酸(10mg/mL)的PBS重悬并作用10min,然后10000g离心20min洗涤，采用10mL含有裂解物(50mg/mL) 的PBS溶液重悬并作用10min,然后在10000g离心20分钟并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h；在粒子使用前将其用7mLPBS重悬然后加入3mL的裂解液组分(蛋白质浓度50mg/mL)并室温作用10min，得到内外都负载裂解物的经冷冻硅化和添加带电物质修饰的纳米粒子系统。该纳米粒子平均粒径为350nm左右，纳米粒子表面电位为-3mV左右；每1mg PLA纳米粒子约负载260μg蛋白质或多肽组分，每1mgPLA纳米粒内负载芬戈莫德为0.03mg。
对照多肽纳米粒子的制备材料和制备方法相同，负载四种质量等量的β细胞多肽抗原，其他与负载全细胞抗原的纳米粒子相同。多肽纳米粒子每1mgPLA纳米粒所负载芬戈莫德为0.03mg，平均粒径为350nm左右，纳米粒子表面电位为-3mV左右。所负载的多肽抗原为InsulinB 9-23,InsulinA 14-20,IGRP206-214,和GAD 225-244。
空白纳米粒粒径为330nm左右，空白纳米粒制备材料和制备方法相同，负载等量佐剂但不负载任何细胞裂解物组分。
(3)DC的制备
处死小鼠后摘取小鼠淋巴结，将小鼠淋巴结剪碎后使用细胞筛网过滤制备淋巴结细胞单细胞悬液。然后使用流式细胞术从淋巴结细胞单细胞悬液中分选CD11c +的DC。
(4)骨髓来源巨噬细胞(BMDM)的制备
将C57小鼠麻醉后脱臼处死，将小鼠使用75％乙醇的消毒，然后用剪刀在小鼠背部剪开一小口，用手直接撕开皮肤至小鼠小腿关节处，去除小鼠足关节以及皮肤。用剪刀沿着小鼠大腿根部大转子将后肢拆下来，去掉肌肉组织后放置在含有75％乙醇的培养皿内浸泡5min，更换新的75％乙醇的培养皿移入超净台中。将乙醇浸泡的腿骨移入冷的PBS浸泡，洗去胫骨、股骨表面的乙醇，此过程可重复3次。将清洗好的股骨、胫骨分开，并用剪刀分别将股骨、胫骨两端剪断，使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出，反复吹洗3次，直至腿骨内看不到明显的红色为止。用5mL移液枪将含有骨髓细胞的培养基反复吹打，使细胞团块分散，然后使用70μm细胞滤器将细胞过筛，转移至15mL离心管内，1500rpm/min离心5min，弃上清，加入红细胞裂解液重悬静置5min后1500rpm/min离心5min，弃上清用冷的配置好的骨髓巨噬细胞诱导培养基(含有15％L929培养基的DMEM高糖培养基)重悬，铺板。将细胞培养过夜，以去除贴壁较快的其他杂细胞如纤维细胞等等。收集未贴壁细胞按实验设计安排种入皿或细胞培养板内。巨噬细胞集落刺激因子(M-CSF)以40ng/mL浓度刺激使骨髓细胞向单核巨噬细胞分化。培养8天，光镜下观察巨噬细胞形态变化。8天后消化收集细胞，用抗小鼠F4/80抗体和抗小鼠CD11b抗体，4℃避光孵育30min后，使用流式细胞术鉴定所诱导成功的巨噬细胞的比例。
(5)抗原提呈细胞的激活
将负载全细胞抗原的纳米粒子(500μg)或多肽纳米粒(500μg)或空白纳米粒(500μg)+游离裂解液与制备的DC(500万个)和BMDM细胞(500万个)在15mLRPMI1640完全培养基中共孵育24小时(37℃，5％CO 2)，孵育体系中含有粒细胞－巨噬细胞集落刺激因子(GM-CSF，2000U/mL)、IL-2(200U/mL)、IL-4(500U/mL)、IL-10(2000U/mL)和PD-L1抗体(10ng/mL)。
(6)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的DC和BMDM，然后使用含有磷酸酶抑制剂和蛋白 酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃下使用组织匀浆机在1500rpm搅拌10分钟机械破坏抗原提呈细胞。然后将样品依次过孔径为30μm、10μm、5μm、2μm、0.45μm的膜过滤后，将滤液在14000g离心35分钟后弃去上清液收集沉淀，将沉淀在含有的4％甘露醇的生理盐水中重悬后冷冻干燥，即得纳米疫苗，纳米疫苗粒径为260纳米。
(7)抗原提呈细胞制备的纳米疫苗用于预防I型糖尿病
同实施例1。
(8)实验结果
如图5所示，PBS或者空白微米粒+细胞裂解物激活的抗原提呈细胞制备的纳米疫苗处理的小鼠在25周后有70％-80％患有I型糖尿病；负载抗原多肽的纳米粒激活的抗原提呈细胞制备的纳米疫苗处理的小鼠在25周后有60％患有糖尿病。在采用负载胰腺组织全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗处理的小鼠在25周后只有约30％小鼠患有I型糖尿病。综上所述，本发明所述的纳米疫苗对I型糖尿病具有良好的预防效果。
实施例5基于抗原提呈细胞的纳米疫苗预防I型糖尿病
本实施例中，首先使用6M盐酸胍裂解β细胞。然后，以PLA为纳米粒骨架材料，以雷公藤为免疫抑制剂制备负载有β细胞全细胞抗原的纳米粒子。使用纳米粒子激活抗原提呈细胞后将抗原提呈细胞细胞膜制备纳米疫苗预防I型糖尿病。
(1)β细胞的裂解
将培养的NIT-1细胞系收集后在400g离心5分钟，然后弃去上清并用PBS洗涤两遍，然后采用6M盐酸胍重悬和裂解NIT-1细胞，NIT-1细胞全细胞抗原裂解并溶于6M盐酸胍后即为制备纳米粒子系统的抗原原料来源。
(2)纳米粒子的制备
本实施例中纳米粒子采用复乳法制备。所采用的纳米粒子制备材料PLA分子量为30KDa，所采用的免疫抑制剂为雷公藤。制备方法如前所述，在制备过程中首先采用复乳法在纳米粒子内部负载NIT-1细胞全细胞抗原和雷公藤，然后将100mg纳米粒子在13000g离心25分钟，然后使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子平均粒径为250nm左右，纳米粒子表面电位为-4mV左右；每1mg PLA纳米粒子约负载90μg蛋白质或多肽组分，负载雷公藤0.02mg。该纳米粒子在小鼠动物实验中作为纳米疫苗3使用。
(3)BMDC的制备
制备方法同实施例1。
(4)抗原提呈细胞的激活
将负载全细胞抗原的微米粒子(500μg)与BMDC(1000万个)在20mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO 2)，孵育体系中含有TGF-β(1000U/mL)、IL-10(2000U/mL)、IL-4(500U/mL)和PD-L1抗体(10ng/mL)。
(5)基于抗原提呈细胞细胞膜的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的BMDC(1000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃使用超声(20W) 处理1分钟，然后使用高压均质机(工作压力5MPa)均质处理1分钟。然后将样品在1000g离心15分钟并收集上清液，然后在6000g离心15分钟后弃去沉淀，收集上清液后在16000g离心90分钟后弃去上清液并收集沉淀，将沉淀重悬后与步骤(2)制备的负载全细胞抗原的纳米粒子(50mg)混合后在10W超声30秒后共孵育15分钟后使用0.45μm的滤膜过滤挤出后收集滤液，，然后将滤液在12000g离心20分钟后弃去上清液收集沉淀，将沉淀在生理盐水中重悬后即得纳米疫苗1，粒径为260nm。
通过在400g离心5分钟收集孵育后的BMDC(1000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃使用超声(20W)处理1分钟，然后使用高压均质机(工作压力5MPa)均质处理1分钟。然后将样品在1000g离心15分钟并收集上清液，然后在6000g离心15分钟后弃去沉淀，收集上清液后在16000g离心90分钟后弃去上清液并收集沉淀，将沉淀使用PBS重悬后即可得只含有被激活的DC细胞膜组分的纳米疫苗2，粒径为130nm。
(6)纳米疫苗用于预防I型糖尿病
本研究对照组是PBS组。选取3周龄雌性NOD小鼠进行本实验。在实验中，每组10只NOD小鼠，从第三周开始每隔7天皮下注射100μg纳米疫苗1、或者100μg纳米疫苗2、或者100μg纳米疫苗3，或者100μL PBS，连续给6周。各组小鼠从第8周开始每天记录小鼠血糖情况。以血糖高于11.0mmol·L-1为糖尿病开始发病。记录不同时间段NOD小鼠糖尿病发病情况。
(7)实验结果
如图6所示，PBS处理的小鼠在25周后有80％患有I型糖尿病；纳米疫苗2处理的小鼠在25周后有40％患有糖尿病；纳米疫苗3处理的小鼠在25周后有40％患有糖尿病；纳米疫苗1处理的小鼠在25周后有20％患有糖尿病。这说明纳米疫苗1优于纳米疫苗2和纳米疫苗3，内部负载癌细胞全细胞抗原和表面负载被激活抗原提呈细胞的细胞膜组分都有利于提高纳米疫苗的功效。纳米疫苗2为使用被纳米粒子3激活的DC细胞膜制备的纳米疫苗，为囊泡状的负载细胞膜组分制备的纳米粒子；而纳米疫苗1为内部负载全细胞抗原，表面复杂抗原提呈细胞细胞膜的实心球表面负载细胞膜的纳米粒子，这说明内部负载全细胞抗原，表面负载膜组分的实心纳米疫苗效果优于只是表面负载抗原的囊泡纳米疫苗。由于被激活的抗原提呈细胞膜表面具有MHC分子与抗原多肽结合后的复合物，所以纳米疫苗1和纳米疫苗2表面都负载有MHC分子与抗原多肽结合后的复合物。具有膜结构的纳米疫苗1既可以直接将
Figure PCTCN2022108962-appb-000001
T细胞诱导成具有负向调节功能的调节性T细胞(T reg)，也可以被抗原提呈细胞吞噬后所负载的抗原被抗原提呈细胞降解提呈后间接激活调节性T细胞(T reg)。综上所述，本发明所述的纳米疫苗对I型糖尿病具有良好的预防效果。
实施例6纳米疫苗用于I型糖尿病的预防
本实施例中，首先使用8M尿素裂解胰岛组织，并溶解胰岛组织裂解物组分。然后，以PLA为纳米粒骨架材料，以环磷酰胺为免疫抑制剂制备负载有全细胞抗原的纳米粒子，使用纳米粒子激活抗原提呈细胞后利用抗原提呈细胞制备纳米疫苗用于预防自身免疫疾病-I型糖尿病。
(1)胰岛组织的裂解
处死小鼠后按照实验流程摘取分离小鼠胰岛组织。将胰岛组织研磨并通过细胞过滤网 后使用适量8M尿素裂解细胞，并使用8M尿素溶解细胞裂解物。以上即为制备纳米粒子系统的抗原原料来源。
(2)纳米粒子系统的制备
本实施例中纳米粒子采用溶剂挥发法制备。所采用的纳米粒子制备材料PLA分子量为20KDa，所采用的免疫抑制剂为环磷酰胺，且裂解物组分和环磷酰胺包载于纳米粒子内部。制备方法如前所述，在制备过程中首先采用复乳法在纳米粒子内部负载裂解物组分和免疫抑制剂，在内部负载抗原裂解组分和免疫抑制剂后，将100mg纳米粒子在12000g离心20分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h，得冻干粉后备用。该纳米粒子平均粒径为280nm左右，纳米粒子表面电位为-3mV左右；每1mg PLA纳米粒子约负载100μg蛋白质或多肽组分，每1mg PLA纳米粒负载环磷酰胺0.04mg。空白纳米粒制备材料和制备方法相同，粒径为280nm左右，空白纳米粒负载等量免疫制剂但是不负载任何裂解物组分。对照纳米粒子负载四种等质量的抗原多肽(Insulin B 9-23,Insulin A 14-20,IGRP 206-214,和GAD 225-244)来代替裂解物组分，其他与负载全细胞抗原的纳米粒子相同，对照纳米粒子每1mgPLA纳米粒所负载的环磷酰胺为0.04mg，粒径为280nm左右，表面电位为-3mV左右，每1mg PLA纳米粒子约负载100μg多肽组分。
(3)DC和B细胞的制备
处死小鼠后摘取小鼠淋巴结，制备小鼠淋巴结单细胞悬液，然后使用流式细胞术从淋巴结细胞单细胞悬液中分选出CD11c +DC和CD19 +B细胞。
(4)抗原提呈细胞的激活
将负载全细胞抗原的纳米粒子(500μg)或多肽纳米粒(500μg)与DC(500万个)和B细胞(500万个)在20mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO 2)，或者将负载全细胞抗原的纳米粒子(500μg)与DC(1000万个)在20mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(1000U/mL)、IL-2(100U/mL)、IL-10(2000U/mL)、TGF-β(2000U/mL)。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的DC(500万个)和B细胞(500万个)或者只收集DC细胞(1000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃使用匀浆机在2000rpm搅拌破坏处理25分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，然后将所得上清液与步骤(2)所制备的相对应的激活抗原提呈细胞的纳米粒子(20mg)混合后在2000RPM搅拌2分钟后使用0.45μm的滤膜过滤挤出后收集滤液，然后将滤液在12000g离心30分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米疫苗，纳米疫苗粒径为300纳米。
(6)纳米疫苗用于I型糖尿病的预防
同实施例1。
(7)实验结果
如图7所示，PBS或者空白微米粒+细胞裂解物激活的抗原提呈细胞制备的纳米疫苗处理的小鼠在25周后有70％-80％患有I型糖尿病；纳米疫苗预防处理的小鼠患有I型糖尿病的比率都明显降低。综上所述，本发明所述的纳米疫苗对I型糖尿病具有良好的预防效 果。而且，负载全细胞抗原的纳米粒激活的DC和B细胞制备的纳米疫苗的预防效果优于负载四种抗原多肽的纳米粒子激活的DC和B细胞制备的纳米疫苗。而且，负载全细胞抗原的纳米粒子激活的DC和B细胞混合制备的纳米疫苗效果优于负载癌细胞全细胞抗原的纳米粒子激活的DC制备的纳米疫苗，说明多种被纳米粒子激活的抗原提呈细胞制备的纳米疫苗效果更好。这可能是因为激活的B细胞内的一部分组分包含进抗原提呈细胞制备的纳米疫苗后可以辅助增强纳米疫苗激活特异性T细胞。
实施例7被激活的抗原提呈细胞制备的纳米疫苗用于治疗I型糖尿病
本实施例以I型糖尿病为模型来说明如何使用被纳米粒子激活的抗原提呈细胞制备纳米疫苗，并使用该类疫苗预防和治疗自身免疫疾病。首先裂解β细胞(NIT-1细胞)以制备全细胞水溶性组分和非水溶性组分，并将水溶性组分和非水溶性组分按质量比1:1混合。然后，以PLA为纳米粒骨架材料，以雷帕霉素和胍立莫司(Gusperimus)为免疫抑制剂制备纳米粒子，并用该纳米粒子体外激活抗原提呈细胞后制备纳米疫苗治疗I型糖尿病。
(1)β细胞的裂解及各组分的收集
收集细胞培养的NIT-1细胞系后，加入适量纯水溶胀细胞后反复冻融5次，并伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以大于5000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分；在所得沉淀部分中加入含有8M尿素和2％精氨酸的水溶液溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为水溶液中可溶。将全细胞水溶性组分和非水溶性组分按质量比1:1混合即为制备纳米粒子的原料来源。
(2)纳米粒子的制备
本实施例中纳米粒采用溶剂挥发法制备。所采用的纳米粒子制备材料PLA分子量为20KDa，所采用的免疫抑制剂为雷帕霉素和胍立莫司，且免疫抑制剂分布于纳米粒子内部。制备方法如前所述，在制备过程中首先采用复乳法在纳米粒子内部负载裂解物混合物和免疫抑制剂，在内部负载裂解物和佐剂后，将100mg纳米粒子在10000g离心20分钟，并使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h。使用前将20mg纳米粒重悬于0.9mL PBS中，并于0.1mL含有裂解物混合物(80mg/mL)的样品室温混合孵育5分钟后即可使用。该纳米粒子平均粒径为280nm左右，纳米粒子表面电位为-3mV左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，每1mgPLGA纳米粒含有雷帕霉素和胍立莫司各0.03mg。
(3)抗原提呈细胞的制备
处死C57BL/6后收集小鼠外周血，从外周血中分离外周血单核细胞(PBMC)，然后使用流式细胞术从PBMC中分选出CD11c +DC。本实施例中同时使用DC和BMDM作为抗原提呈细胞。BMDM制备方法同实施例4。
(4)抗原提呈细胞的激活
将负载细胞全细胞抗原的纳米粒子(1000μg)与外周血来源的DC(500万个)和BMDM(500万个)在20mLRPMI1640完全培养基中共孵育48小时(37℃，5％CO 2)，或者将负载癌细胞全细胞抗原的纳米粒子(500μg)与外周血来源的DC(1000万个)在RPMI1640完全培养基中共孵育72小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(1000U/mL)、IL-2(100U/mL)、IL-10(2000U/mL)、IL-4(500U/mL)。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的外周血来源的DC(500万个)及BMDM(500万个) 或者只收集外周血来源的DC(1000万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在高压均质机中(5000bar)中处理5分钟。然后将样品在2000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，将上清液与步骤(2)所制备的相对应的纳米粒子(60mg)常温共孵育1小时后使用0.45μm的滤膜过滤挤出后收集滤液，然后在13000g离心20分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米疫苗，纳米疫苗粒径为320纳米。
(6)纳米疫苗用于I型糖尿病的治疗
选取25周龄已经糖尿病发病的雌性NOD小鼠进行本实验。在实验中，每组10只NOD小鼠。从第0天开始每只小鼠每隔3天皮下注射100μg纳米疫苗或PBS，连续给6次。各组小鼠每天记录小鼠血糖情况。以血糖高于11.0mmol·L-1为糖尿病发病，以血糖连续三天低于11.0mmol·L-1为糖尿病缓解治愈。记录不同时间段NOD小鼠糖尿病发病情况。
(7)实验结果
如图8所示，PBS对照组小鼠的血糖始终高于正常。而纳米疫苗治疗组部分小鼠I型糖尿病痊愈。而且，使用激活的DC和巨噬细胞混合制备的纳米疫苗效果优于纳米粒子激活的DC制备的纳米疫苗，说明多种被纳米粒子激活的抗原提呈细胞制备的纳米疫苗效果更好。综上所述，本发明所述抗原提呈细胞制备的纳米疫苗对于I型糖尿病具有良好的治疗效果。
实施例8激活的抗原提呈细胞制备的纳米疫苗用于治疗I型糖尿病
本实施例以I型糖尿病为模型来说明如何使用负载β细胞全细胞抗原的纳米粒子激活抗原提呈细胞后，使用抗原提呈细胞制备的纳米疫苗治疗I型糖尿病。
(1)β细胞的的裂解及各组分的收集
收集培养的NIT-1细胞，将NIT-1细胞使用8M尿素水溶液(含500mM氯化钠)中裂解后用8M尿素水溶液溶解裂解物组分即为制备纳米粒子的抗原来源。
(2)纳米粒子的制备
本实施例中纳米粒子采用复乳法制备。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的抑制剂为雷帕霉素和依维莫司。在内部负载裂解组分和免疫抑制剂后，将100mg纳米粒子在10000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子平均粒径为360nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分，每1mg PLGA纳米粒负载雷帕霉素和依维莫司各0.02mg。
空白纳米粒的制备材料和制备方法同上，粒径为350nm左右，只负载等量佐剂但是不负载任何裂解物组分。
(3)DC的制备
DC为来源于外周血的DC和BMDC的混合DC。BMDC制备方法同上。处死C57BL/6后收集小鼠外周血，从外周血中分离外周血单核细胞(PBMC)，然后使用流式细胞术从PBMC中分选出CD11c +DC。
(4)抗原提呈细胞的激活
将负载全细胞抗原的纳米粒子(800μg)或者空白纳米粒子(800μg)+等量游离裂解液与外周血来源的DC(800万个)和BMDC(800万个)在15mL高糖DMEM完全培养基 中共孵育48小时(37℃，5％CO 2)，孵育体系中含有IL-2(100U/mL)、IL-10(2000U/mL)、IL-4(1000U/mL)和TGF-β(1000U/mL)。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的外周血来源的DC(800万个)和BMDC(800万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声1分钟后使用匀浆机在1000rpm处理3分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液。将上清液与实施例1中步骤2所制备的相应的纳米粒子(50mg)以及DSPE-PEG-CD32单抗在10W超声处理2分钟后使用0.45μm的滤膜过滤挤出后收集滤液，然后将滤液在15000g离心30分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米疫苗1。或者将上清液与实施例1中步骤2所制备的相应的纳米粒子(50mg)在10W超声处理2分钟后使用0.45μm的滤膜过滤挤出后收集滤液，然后将滤液在15000g离心30分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米疫苗2。纳米疫苗1和纳米疫苗2的粒径都为380纳米。
(6)纳米疫苗用于I型糖尿病的治疗
同实施例7。
(7)实验结果
如图9所示，PBS对照和空白纳米粒激活的抗原提呈细胞制备的纳米疫苗处理的小鼠的血糖始终高于正常。而负载全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗治疗组部分小鼠I型糖尿病痊愈。而且，带有CD32单抗靶头的纳米疫苗效果好于不带靶头的纳米疫苗。综上所述，本发明所述抗原提呈细胞制备的纳米疫苗对于I型糖尿病具有良好的治疗效果。本实施例纳米疫苗中使用CD32单克隆抗体作为主动靶向的靶头，在实际应用中也可以使用甘露糖、甘露聚糖、CD205单抗、CD19单抗等任何具有靶向靶细胞能力的靶头。
实施例9基于抗原提呈细胞的纳米疫苗用于I型糖尿病的预防
本实施例以小鼠胰腺组织来说明如何制备负载有β细胞的胰腺组织全细胞抗原的微米粒子，然后使用该微米粒子激活抗原提呈细胞后将抗原提呈细胞制备成纳米疫苗，并应用该纳米疫苗预防I型糖尿病。胰腺部位含有小鼠β细胞，可以用来作为胰岛β细胞来源制备疫苗。首先摘取小鼠的胰腺组织后分别制备所得组织的全细胞的水溶性组分和非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以编码TGF-β的mRNA和雷帕霉素为免疫抑制剂采用溶剂挥发法制备负载全细胞抗原的微米粒子，并用该微米粒子激活的抗原提呈细胞制备纳米疫苗。
(1)胰腺组织全细胞抗原的收集
将BALB/c小鼠处死后摘取小鼠的胰腺组织。将胰腺组织切块后研磨，通过细胞过滤网加入适量纯水并反复冻融和超声处理至少8次。待组织细胞裂解后，将组织细胞裂解物使用紫外线照射15分钟后，将裂解物置于95℃加热处理10分钟，然后将组织的裂解物以大于3000RPM的转速离心5min取上清液即为组织细胞中可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的原非水溶性组分转化为在8M尿素水溶液中可溶。将水溶性组分和溶解于8M尿素中的原非水溶性组分按照 质量比1:1混合，即为制备激活抗原提呈细胞的的微米粒子的抗原来源。
(2)用于激活抗原提呈细胞的微米粒子的制备
本实施例中制备微米粒子采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫抑制剂为编码TGF-β的mRNA和雷帕霉素，编码TGF-β的mRNA和雷帕霉素分布于微米粒子内部，在制备时mRNA溶解于水相中而雷帕霉素溶解于有机相中。制备方法如前所述，微米粒子平均粒径为1.5μm左右，表面电位Zeta potential为-8mV左右，每1mg PLGA微米粒子约负载90μg蛋白质或多肽组分，每1mgPLGA微米粒子负载mRNA为0.01mg。空白微米粒子粒径为1.5μm左右，空白微米粒负载等量mRNA但不负载任何裂解物组分。
(3)BMDC和B细胞的制备
BMDC的制备同实施例1。来源于小鼠外周血的B细胞的制备方法同上。
(4)抗原提呈细胞的激活
将负载细胞全细胞抗原的微米粒子(1000μg)或空白微米粒子(1000μg)与BMDC(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)；或者将负载全细胞抗原的微米粒子(1000μg)与B细胞(1000万个)在15mL高糖DMEM完全培养基中共孵育72小时(37℃，5％CO2)；孵育体系中含有IL-10(2000U/mL)、IL-2(500U/mL)、IL-4(200U/mL)、IL-13(200U/mL)。
(5)基于抗原提呈细胞的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的DC或者B细胞，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(10W)超声10分钟。然后将样品在依次过孔径为10μm、5μm、2μm、1μm和0.45μm的膜过滤后收集滤液，然后在14000g离心40分钟后弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米疫苗，纳米疫苗粒径为160nm。
(6)纳米疫苗用于I型糖尿病的预防
同实施例1。
(7)实验结果
如图10所示，与PBS和空白微米粒+细胞裂解物激活的抗原提呈细胞制备的纳米疫苗处理的小鼠相比，负载全细胞抗原的微米粒子激活的抗原提呈细胞制备的纳米疫苗处理的小鼠患有I型糖尿病的比例明显降低。而且，微米粒激活的DC制备的纳米疫苗效果好于微米粒激活的B细胞制备的纳米疫苗。由此可见，本发明所述的纳米疫苗对I型糖尿病具有预防效果，不同抗原提呈细胞制备的纳米疫苗效果有差异。
实施例10激活的抗原提呈细胞制备的纳米疫苗用于预防I型糖尿病
本实施例以I型糖尿病为模型来说明如何使用负载β细胞全细胞抗原的纳米粒子激活抗原提呈细胞后，使用抗原提呈细胞制备的纳米疫苗预防I型糖尿病。而且，在β细胞裂解之前对其进行适当的处理，可以增加抗原的含量，进而提高负载抗原的粒子激活抗原提呈细 胞的效率。
(1)β细胞的的裂解及各组分的收集
收集培养的NIT-1细胞，离心后去除培养基，用超纯水重悬NIT-1细胞，然后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融8次，并伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以2000g的转速离心15分钟并取上清液即为NIT-1细胞可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素(含500mM氯化钠)水溶液溶解沉淀部分即可将NIT-1细胞中不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。将上述水溶性组分和非水溶性组分按质量比1:1混合即为制备对照纳米粒子的抗原来源。
在高糖培养基中将葡萄糖含量增加一倍，并同时加入0.05μM/L毒胡萝卜素(Thapsigargin)至培养基中孵育培养NIT-1细胞12小时。增加葡萄糖的含量或添加毒胡萝卜素都可以增加β细胞中抗原的含量。尔后，收集所培养的NIT-1细胞，离心后去除培养基，用超纯水重悬NIT-1细胞，然后加一定量超纯水后反复冻融8次，并伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以2000g的转速离心15分钟并取上清液即为NIT-1细胞可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素(含500mM氯化钠)水溶液溶解沉淀部分即可将NIT-1细胞中不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。将上述水溶性组分和非水溶性组分按质量比1:1混合即为制备激活抗原提呈细胞的纳米粒子的抗原来源。
(2)纳米粒子的制备
本实施例中纳米粒子和对照纳米粒子采用复乳法制备。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的抑制剂为雷帕霉素和依维莫司。在内部负载裂解组分和免疫抑制剂后，将100mg纳米粒子在12000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子平均粒径为260nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分，每1mg PLGA纳米粒负载雷帕霉素和依维莫司各0.02mg。
(3)DC的制备
DC为来源于外周血的DC和BMDC的混合DC。二者的制备方法同上。
(4)抗原提呈细胞的激活
将负载全细胞抗原的纳米粒子(800μg)与外周血来源的DC(800万个)和BMDC(800万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)，孵育体系中含有IL-13(500U/mL)、IL-10(2000U/mL)、IL-4(1000U/mL)和TGF-β(2000U/mL)。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的外周血来源的DC(800万个)和BMDC(800万个)，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声1分钟后使用匀浆机在1000rpm处理3分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液在8000g离心15分钟后收集上清液，将上清液使用0.22μm的滤膜过滤挤出后收集滤液，将滤液与实施例1中步骤2所制备的相应的纳米粒子(30mg)以及DSPE-PEG-CD32单抗(30μg)在1200RPM搅拌处理2分钟后使用0.45μm的滤膜过滤挤出后收集滤液，然后将滤液在15000g离心30分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后即得纳米疫苗，纳米疫苗粒径为280纳米。
(6)纳米疫苗用于I型糖尿病的预防
同实施例1。
(7)实验结果
如图11所示，与PBS对照组的小鼠相比，负载全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗处理的小鼠患有I型糖尿病的比例明显降低。而且，使用经过高糖和添加化学物质处理的β细胞制备的纳米粒激活的抗原提呈细胞所制备的纳米疫苗效果好于为做上述处理的β细胞制备的纳米粒激活的抗原提呈细胞制备的纳米疫苗。由此可见，本发明所述的纳米疫苗对I型糖尿病具有预防效果，而且，对β细胞进行适当处理增加抗原含量有助于提高被激活的抗原提呈细胞制备的纳米疫苗的疗效。
实施例11激活的抗原提呈细胞制备的纳米疫苗用于预防I型糖尿病
(1)β细胞的的裂解及各组分的收集
收集培养的NIT-1细胞，离心后去除培养基，用超纯水重悬NIT-1细胞，然后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融8次，并伴有超声以破坏裂解细胞。待细胞裂解后，加入核酸酶(2mg/mL)后在37℃作用30分钟，然后在95℃作用5分钟灭活核酸酶，将裂解物以大于100g的转速离心1分钟以上并取上清液即为NIT-1细胞可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素(含500mM氯化钠)水溶液溶解沉淀部分即可将NIT-1细胞中不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。将上述水溶性组分和非水溶性组分按质量比1:1混合即为制备对照纳米粒子的抗原来源。
(2)纳米粒子的制备
本实施例中纳米粒子和对照纳米粒子采用复乳法制备。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的抑制剂为雷帕霉素和他克莫司。在内部负载裂解组分和免疫抑制剂后，将100mg纳米粒子在12000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子平均粒径为250nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分，每1mg PLGA纳米粒负载雷帕霉素和他克莫司各0.02mg。
(3)DC的制备
DC为来源于外周血的DC和BMDC的混合DC。二者的制备方法同上。
(4)抗原提呈细胞的激活
将负载全细胞抗原的纳米粒子(800μg)与外周血来源的DC(1000万个)和BMDC(1000万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)，孵育体系中含有IL-13(500U/mL)、IL-10(2000U/mL)、IL-4(1000U/mL)和TGF-β(2000U/mL)。或者将负载全细胞抗原的纳米粒子(800μg)与外周血来源的DC(800万个)和BMDC(800万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)，但是孵育体系中不加入任何细胞因子。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的BMDC或者BMDM，然后使用含有0.0759M蔗糖和0.225M甘露醇的30mM pH 7.0 Tris-HCl缓冲液中1200rpm 3min离心清洗三次，然后在磷酸酶抑制剂和蛋白酶抑制剂的存在下超声机械破坏抗原提呈细胞。经过离心后所获细胞膜用10mM pH 7.5的Tris-HCl和1mM EDTA的溶液清洗。然后将样品依次过孔径为30μm、 10μm、5μm、2μm、0.45μm的膜过滤后，将滤液在12000g离心25分钟后弃去上清液收集沉淀，将沉淀在含有的4％甘露醇的生理盐水中重悬后冷冻干燥，即得纳米疫苗，纳米疫苗粒径为260纳米。
(6)纳米疫苗用于I型糖尿病的预防
同实施例1。
(7)实验结果
如图12所示，与PBS对照组的小鼠相比，负载全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗处理的小鼠患有I型糖尿病的比例明显降低。而且，在纳米粒激活的抗原提呈细胞过程中加入细胞因子或抗体激活的抗原提呈细胞所制备的纳米疫苗效果好于在纳米粒激活的抗原提呈细胞过程中未加入任何细胞因子或抗体的抗原提呈细胞制备的纳米疫苗。由此可见，本发明所述的纳米疫苗对I型糖尿病具有预防效果，而且，在抗原提呈细胞激活过程中细胞因子和/或抗体的添加有助于提高被激活的抗原提呈细胞制备的纳米疫苗的疗效。
实施例12激活的抗原提呈细胞制备的纳米疫苗用于预防I型糖尿病
(1)β细胞的的裂解及各组分的收集
收集培养的NIT-1细胞，离心后去除培养基，用超纯水重悬NIT-1细胞，然后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融8次，并伴有超声以破坏裂解细胞。待细胞裂解后，加入核酸酶(2mg/mL)后在37℃作用30分钟，然后在95℃作用5分钟灭活核酸酶，将裂解物以大于100g的转速离心1分钟以上并取上清液即为NIT-1细胞可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素(含500mM氯化钠)水溶液溶解沉淀部分即可将NIT-1细胞中不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。将上述水溶性组分和非水溶性组分按质量比1:1混合即为制备对照纳米粒子的抗原来源。
(2)纳米粒子的制备
本实施例中纳米粒子和对照纳米粒子采用复乳法制备。所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的抑制剂为雷帕霉素和白介素2，使用KALA多肽(WEAKLAKALAKALAKHLAKALAKALKACEA)作为溶酶体逃逸物质。在内部负载裂解组分、溶酶体逃逸物质和免疫抑制剂后，将100mg纳米粒子在12000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后冷冻干燥48h后备用。该纳米粒子平均粒径为250nm左右，纳米粒子表面电位为-5mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分，每1mg PLGA纳米粒负载雷帕霉素和白介素2各0.02mg，负载KALA多肽0.03mg。对照纳米粒子制备材料和制备方法相同，平均粒径为250nm左右，表面电位为-5mV左右；每1mg PLGA纳米粒子约负载80μg蛋白质和多肽组分，每1mg PLGA纳米粒负载雷帕霉素和白介素2各0.03mg，不负载任何增加溶酶体逃逸的物质。
(3)DC的制备
DC为来源于外周血的DC和BMDC的混合DC。二者的制备方法同上。
(4)抗原提呈细胞的激活
将负载全细胞抗原的纳米粒子(800μg)与外周血来源的DC(800万个)和BMDC(800万个)在15mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO2)，孵育体系中含有IL-13(500U/mL)、IL-10(2000U/mL)、IL-4(1000U/mL)和TGF-β (2000U/mL)。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的BMDC或者BMDM，然后使用含有0.0759M蔗糖和0.225M甘露醇的30mM pH 7.0 Tris-HCl缓冲液中1200rpm 3min离心清洗三次，然后在磷酸酶抑制剂和蛋白酶抑制剂的存在下超声机械破坏抗原提呈细胞。经过离心后所获细胞膜用10mM pH 7.5的Tris-HCl和1mM EDTA的溶液清洗。然后将样品依次过孔径为30μm、10μm、5μm、2μm、0.45μm的膜过滤后，将滤液在12000g离心25分钟后弃去上清液收集沉淀，将沉淀在含有的4％甘露醇的生理盐水中重悬后冷冻干燥，即得纳米疫苗，纳米疫苗粒径为260纳米。
(6)纳米疫苗用于I型糖尿病的预防
同实施例1。
(7)实验结果
如图13所示，与PBS对照组的小鼠相比，负载全细胞抗原的纳米粒子激活的抗原提呈细胞制备的纳米疫苗处理的小鼠患有I型糖尿病的比例明显降低。而且，负载KALA多肽的纳米粒激活的抗原提呈细胞所制备的纳米疫苗效果好于未负载KALA多肽的纳米粒激活的抗原提呈细胞制备的纳米疫苗。由此可见，本发明所述的纳米疫苗对I型糖尿病具有预防效果，而且，在激活抗原提呈细胞的纳米粒子或微米粒子中加入KALA多肽有助于提高被激活的抗原提呈细胞制备的纳米疫苗的疗效。本实施例中加入的为KALA多肽，在实际使用过程中也可以负载其他具有增加溶酶体逃逸功能的物质的中的一种或多种，比如精氨酸、聚精氨酸、赖氨酸、聚赖氨酸、组氨酸、聚组氨酸、NH 4HCO 3、带有正电荷的多肽、鱼精蛋白、组蛋白等。
实施例13纳米疫苗预防自身免疫疾病
(1)β细胞的裂解
在高糖培养基中将葡萄糖含量增加一倍，并同时加入0.05μM/L毒胡萝卜素(Thapsigargin)至培养基中孵育培养NIT-1细胞12小时。增加葡萄糖的含量和添加毒胡萝卜素可以增加β细胞中抗原的含量。尔后，收集所培养的NIT-1细胞，离心后去除培养基，用10％脱氧胆酸钠水溶液(含8M精氨酸)重悬和裂解NIT-1细胞，将裂解物组分以10％脱氧胆酸钠水溶液(含8M精氨酸)溶解即为制备激活抗原提呈细胞的纳米粒子的抗原来源。
(2)纳米粒子的制备
本实施例中纳米粒子采用溶剂挥发法制备，所采用的纳米粒子制备材料PLGA分子量为38KDa-54KDa，所采用免疫抑制剂为雷帕霉素和霉酚酸酯，所采用的增加溶酶体逃逸的物质为聚精氨酸和RALA多肽，免疫抑制剂、聚精氨酸和RALA多肽均负载于纳米粒子内。制备方法如前所述，在制备过程中首先采用复乳法在纳米粒子内部裂解组分、免疫抑制剂、聚精氨酸和RALA多肽，然后将100mg PLGA纳米粒子在13000g离心20min后将沉淀使用4％海藻糖重悬后冷冻干燥48小时后备用。该纳米粒子平均粒径为260nm左右；每1mg PLGA纳米粒子约负载140μg蛋白质或多肽组分，负载雷帕霉素和霉酚酸酯各0.03mg，负载聚精氨酸和RALA多肽各0.02mg。
空白纳米粒子制备材料和制备方法相同，空白纳米粒子平均粒径为260nm左右，每1mg PLGA空白纳米粒子负载雷帕霉素和霉酚酸酯各0.03mg，负载聚精氨酸和RALA多肽各 0.02mg，但是不负载任何裂解物组分。
(3)抗原提呈细胞的激活
本实施例使用DC2.4细胞、来源于脾细胞的B细胞和BMDM作为混合抗原提呈细胞，三种抗原提呈细胞的制备方法同上。将DC2.4细胞、B细胞和BMDM按数量比1:1:1混合。将1mg负载全细胞抗原的纳米粒子与3000万个混合抗原提呈细胞(1000万个DC+1000万个B细胞+1000万个BMDM)在25mL高糖DMEM完全培养基中共孵育48小时(37℃，5％CO 2)，孵育体系中含有IL-7(100U/mL)、IL-10(1000U/mL)、IL-4(500U/mL)和TGF-β(1000U/mL)。孵育完成后在400g离心5分钟收集细胞沉淀，用PBS离心洗涤两遍后收集混合抗原提呈细胞备用。被激活后的混合抗原提呈细胞细胞作为活细胞疫苗3。
(4)纳米疫苗的制备
在高糖培养基中将葡萄糖含量增加一倍，并同时加入0.05μM/L毒胡萝卜素(Thapsigargin)至培养基中孵育培养NIT-1细胞18小时。尔后，收集所培养的NIT-1细胞。将3000万个NIT-1细胞与3000万个步骤(3)激活的混合抗原提呈细胞(1000万个DC+1000万个B细胞+1000万个BMDM)混合，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤混合细胞两遍，将细胞重悬在PBS水中后在4℃低功率(10W)超声20分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液依次过孔径为30μm、10μm、5μm、2μm、1μm、0.45μm、0.22μm的膜过滤后，收集滤液，然后将滤液在18000g离心50分钟后收集弃去上清液收集沉淀，将沉淀在PBS中重悬后得到混合细胞膜组分。然后将混合细胞膜组分与步骤(2)制备的负载全细胞组分的纳米粒子(100mg)或者空白纳米粒子(100mg)在1500RPM搅拌5分钟，尔后在室温共孵育15分钟后使用0.45μm的滤膜过滤挤出，然后在13000g离心25分钟后使用10mL的冻干保护剂(含2％海藻糖+2％甘露醇+1％蔗糖)水溶液重悬，尔后冷冻干燥48小时后即得纳米疫苗。其中，使用负载全细胞组分的纳米粒子与混合细胞膜组分共作用得到的纳米疫苗为纳米疫苗1，纳米疫苗1粒径为270纳米；使用空白纳米粒子与混合细胞膜组分共作用得到的纳米疫苗为纳米疫苗2，粒径为270纳米。
(5)纳米疫苗用于I型糖尿病的预防
本研究对照组是PBS组。选取3周龄雌性NOD小鼠进行本实验。在实验中，每组10只NOD小鼠,从第三周开始每隔7天皮下注射100μg纳米疫苗1、或者100μg纳米疫苗2、或者300万个混合抗原提呈细胞疫苗3(100万个DC+100万个B细胞+100万个BMDM)，或者100μL PBS，连续给6周。各组小鼠从第8周开始每天记录小鼠血糖情况。以血糖高于11.0mmol·L-1为糖尿病开始发病。记录不同时间段NOD小鼠糖尿病发病情况。
(6)抗原特异性调节性T细胞(T reg)的分析
选取6-8周的雌性C57BL/6为模型小鼠,在第0天、第7天、第14天、第28天和第42天分别每只小鼠皮下注射100μg纳米疫苗1、或者纳米疫苗2、或者300万个混合抗原提呈细胞疫苗3(100万个DC+100万个B细胞+100万个BMDM)、或者PBS。在第45天处死小鼠，摘取小鼠脾脏并制备脾细胞单细胞悬液，使用磁珠分选法从小鼠脾细胞中分选出B细胞和T细胞。将100μg负载β细胞全细胞抗原的纳米粒子、500万个B细胞和100万个T细胞在5mL RPMI1640完全培养基中共孵育48小时(37℃,5％CO 2)。然后收集孵育后细胞并标记活死细胞染料、CD3抗体、CD8抗体、CD4抗体、CD25抗体、Ly49抗体和FOXP3抗体，尔后流式细胞术分析T细胞亚群中CD4 +CD25 +FOXP3 +T细胞占CD4 +T细胞的比例和 CD8+Ly49 +T占CD8 +T细胞的比例。
(7)实验结果
如图14a所示，与PBS组相比，疫苗处理组的小鼠患I型糖尿病的比例都明显降低。而且，纳米疫苗1效果明显好于纳米疫苗2和活细胞疫苗3。这说明内部负载全细胞抗原同时表面负载含有抗原的细胞和被激活的抗原提呈细胞混合细胞膜组分的纳米疫苗好于内部不负载抗原仅表面负载混合细胞膜组分的纳米疫苗，也说明内部只负载免疫抑制剂和溶酶体逃逸物质，同时表面负载含有抗原的细胞和被激活的抗原提呈细胞混合细胞膜组分的纳米疫苗，也好于被负载全细胞抗原的纳米粒子激活的混合抗原提呈细胞活细胞疫苗。综上所述，说明本发明所述的内部负载全细胞抗原表面负载含有混合细胞细胞膜组分的纳米疫苗对I型糖尿病具有很好的预防效果。
如图14b和14c所示，纳米疫苗1所能诱导的自身抗原特异性T reg多于纳米疫苗2和纳米疫苗3。这说明内部负载细胞全细胞抗原抗原同时表面负载含有混合细胞的细胞膜有利于诱导产生更多的自身抗原特异性T reg。因为可以抑制具有细胞杀伤能力的T eff，所以所诱导的就能对预防或治疗I型糖尿病有疗效。
本实施例中，纳米疫苗表面负载的是含有自身抗原细胞的细胞膜膜组分，在实际应用中，也可以使用含有自身抗原的细胞分泌的细胞外囊泡的膜组分。
实施例14纳米疫苗用于I型糖尿病的治疗
(1)β细胞的裂解
将培养的NIT-1细胞在400g离心5分钟，然后用PBS洗涤两遍后重悬于超纯水中。所得细胞分别采用紫外线和高温加热进行灭活和变性处理，然后加入超纯水并反复冻融5次辅以超声裂解细胞，然后加入核酸酶作用5分钟，再在95℃作用10分钟灭活核酸酶。将细胞裂解物在5000g离心10分钟，上清液即为水溶性组分，将沉淀物使用10％辛基葡萄糖苷溶解后即为溶解后的原非水溶性抗原，将水溶性组分和非水溶性组分按质量比2:1混合，即为制备微米粒子所需的裂解物组分。
(2)微米粒子系统的制备
本实施例中制备微米粒子系统及作为对照微米粒子采用复乳法，微米粒子骨架材料PLGA分子量为38KDa-54KDa，所采用的免疫抑制剂为甲基强的松龙，所采用的溶酶体逃逸增加物质为NH 4HCO 3和蜂毒肽。制备时先采用复乳法制备内部负载裂解物组分、甲基强的松龙、NH 4HCO 3和蜂毒肽的微米粒子,然后将100mg微米粒子在9000g离心20分钟，使用10mL含4％海藻糖的超纯水重悬后干燥48h后备用。该微米粒子平均粒径为3.1μm左右，表面电位为-7mV左右；每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分，含甲基强的松龙0.01mg，含NH4HCO3和蜂毒肽各0.02mg。对照微米粒子制备材料和制备方法与如上方法相同，对照微米粒子平均粒径为3.1μm左右，表面电位为-7mV左右；每1mg PLGA微米粒子约负载110μg蛋白质或多肽组分，不负载任何免疫抑制剂，只负载NH 4HCO 3和蜂毒肽各0.02mg。
(3)抗原提呈细胞的制备
本实施例使用DC2.4细胞系作为抗原提呈细胞。
(4)抗原提呈细胞的激活
将负载癌细胞全细胞抗原的微米粒子(1000μg)与DC2.4(1000万个)在15mL高糖 DMEM完全培养基中共孵育48小时(37℃，5％CO 2)；孵育体系中含有GM-CSF(2000U/mL)、IL-2(100U/mL)、IL-10(2000U/mL)、IL-13(200U/mL)。
(5)抗原提呈细胞来源的纳米疫苗的制备
通过在400g离心5分钟收集孵育后的DC，然后使用含有蛋白酶抑制剂的4℃磷酸盐缓冲溶液(PBS)洗涤细胞两遍，将细胞重悬在PBS水中后在4℃低功率(20W)超声2分钟。然后将样品在3000g离心15分钟并收集上清液，将上清液依次通过30μm,10μm,5μm,1μm,0.45μm的膜过滤，然后在15000g离心60分钟即得纳米疫苗，纳米疫苗粒径平均为250纳米。
(5)纳米疫苗治疗I型糖尿病
同实施例7。
(6)实验结果
如图15所示，与PBS对照组相比，微米粒子激活的抗原提呈细胞制备的纳米疫苗处理的小鼠I型糖尿病小鼠经治疗后得到改善。而且，同时负载免疫抑制剂和溶酶体逃逸物质的微米粒子激活的抗原提呈细胞制备的纳米疫苗优于只负载溶酶体逃逸物质而不负载免疫抑制剂的微米粒子激活的抗原提呈细胞制备的纳米疫苗。说明免疫抑制剂能够提高微米粒子激活抗原提呈细胞的效率，有利于使抗原提呈细胞制备的纳米疫苗更好的诱导T细胞转变成具有特异性免疫抑制功能的调节性T细胞。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法，其特征在于，包括以下步骤：
    S1、将抗原提呈细胞与负载自身免疫疾病全细胞抗原的第一递送粒子共孵育，得到预激活的抗原提呈细胞；
    S2、将预激活的抗原提呈细胞细胞膜制备成纳米囊泡，得到所述自身免疫疾病疫苗；
    或将预激活的抗原提呈细胞的细胞膜组分负载在负载自身免疫疾病全细胞抗原的第二递送粒子上，得到所述自身免疫疾病疫苗；
    其中，
    所述第一递送粒子或第二递送粒子分别独立地为纳米粒子或微米粒子；
    所述自身免疫疾病全细胞抗原由以下步骤制备得到：将含有自身免疫疾病抗原的细胞或组织冷冻，加水进行冻融裂解，收集上清液以及沉淀中经溶解剂溶解后转为可溶的部分，得到所述自身免疫疾病全细胞抗原；或将含有自身免疫疾病抗原的细胞或组织加溶解剂进行裂解，收集可溶部分，得到所述自身免疫疾病全细胞抗原。
  2. 根据权利要求1所述的制备方法，其特征在于：将所述预激活的抗原提呈细胞的细胞膜组分负载在负载自身免疫疾病全细胞抗原的第二递送粒子上的同时，还包括将含有自身免疫疾病抗原细胞的细胞膜组分负载在所述第二递送粒子上。
  3. 根据权利要求1所述的制备方法，其特征在于：在步骤S1中，共孵育时孵育体系中含有细胞因子和/或抗体；所述细胞因子选自白介素1受体拮抗剂、生长因子、白介素、干扰素、肿瘤坏死因子、集落刺激因子、活化素和抑制素中的一种或多种；所述抗体选自PD1抗体、PD-L1抗体、CTAL-4抗体、TIGIT抗体、TIM-3抗体、LAG-3抗体、αCD-8抗体、αCD-28抗体、αCD-40抗体、αOX-40抗体和αOX-40L抗体中的一种或多种。
  4. 根据权利要求1所述的制备方法，其特征在于：所述溶解剂选自尿素、盐酸胍、脱氧胆酸盐、十二烷基硫酸盐、甘油、蛋白质降解酶、白蛋白、卵磷脂、无机盐、Triton、吐温、氨基酸、糖苷和胆碱中的一种或多种。
  5. 根据权利要求1所述的制备方法，其特征在于：在步骤S1中，共孵育时孵育体系中含有葡萄糖和/或毒胡萝卜素。
  6. 根据权利要求1所述的制备方法，其特征在于：所述抗原提呈细胞选自树突状细胞、B细胞和巨噬细胞中的至少一种。
  7. 根据权利要求1所述的制备方法，其特征在于：所述第一递送粒子或第二递送粒子上负载有抑制免疫的物质；所述抑制免疫的物质选自mRNA、DNA、糖皮质激素类药、钙调磷酸酶抑制药、抗代谢药、抗体、细胞因子、烷化剂、环孢素、雷帕霉素、他克莫司、胍立莫司、芬戈莫德、甲基强的松龙、雷公藤、霉酚酸酯、环磷酰胺、硫唑嘌呤、依维莫司、山地明、赛斯平、环孢多肽A、新出地明、TGF-β、白介素、人参和黄芪中的一种或多种。
  8. 根据权利要求1所述的制备方法，其特征在于：所述第一递送粒子或第二递送粒子上负载有KALA多肽、RALA多肽、蜂毒肽、精氨酸、聚精氨酸、赖氨酸、聚赖氨酸、组氨酸、聚组氨酸、NH 4HCO 3、鱼精蛋白和组蛋白中的一种或多种。
  9. 权利要求1-8任一项所述的制备方法制备得到的自身免疫疾病疫苗。
  10. 权利要求9所述的自身免疫疾病疫苗在制备用于治疗或预防自身免疫疾病药物中的应用。
PCT/CN2022/108962 2022-06-10 2022-07-29 一种来源于预激活抗原提呈细胞的自身免疫疾病疫苗的制备方法及其应用 WO2023236331A1 (zh)

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