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WO2021073659A2 - Vaccine vector prepared on basis of anionic polymers and derivatives thereof - Google Patents

Vaccine vector prepared on basis of anionic polymers and derivatives thereof Download PDF

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WO2021073659A2
WO2021073659A2 PCT/CN2020/136150 CN2020136150W WO2021073659A2 WO 2021073659 A2 WO2021073659 A2 WO 2021073659A2 CN 2020136150 W CN2020136150 W CN 2020136150W WO 2021073659 A2 WO2021073659 A2 WO 2021073659A2
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antigen
anionic polymer
virus
sulfate
derivatives
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French (fr)
Chinese (zh)
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WO2021073659A3 (en
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孙逊
张志荣
龚涛
侯盈盈
白淑婷
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四川大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds

Definitions

  • the invention relates to the technical field of medicine, in particular to a vaccine carrier based on anionic polymer and its derivative materials and a preparation method thereof.
  • Vaccination is one of the great victories of modern medicine. It is also an effective means of preventing and eradicating most diseases. It is used to control a variety of infectious diseases and even eradicate smallpox.
  • General vaccines are mainly attenuated or inactivated vaccines, which have high immunogenicity but low safety.
  • the current research is mainly focused on subunit vaccines because they are more pure and safe.
  • the increase in safety is accompanied by a decrease in immunogenicity. Therefore, adjuvants have become a vital component in vaccines to enhance the lasting and effective immune effect of subunit vaccines.
  • the use of vaccine delivery vehicles can induce an effective immune response and provide improved stability, safety, and cost and effectiveness.
  • nano-scale ( ⁇ 1000nm) materials such as virus-like particles, liposomes, ISCOMS, polymers and non-degradable nanospheres, nanoparticles, etc.
  • This kind of carrier can stabilize the vaccine antigen and avoid the degradation of the antigen. It also has a certain adjuvant effect, which can help the antigen to achieve its immune response.
  • the size of the vaccine delivery vehicle will affect its distribution and ultimately affect the antigen response.
  • particles with a size of 20-100nm can pass through the extracellular matrix and directly enter the lymphatic vessels. Larger particles are generally taken up by DC cells under the skin and then migrate to the lymph nodes. The speed of the latter and the amount of antigen delivered are much lower than the former.
  • the nanoparticles can be directly targeted to the lymph nodes and captured by a large number of immune cells (such as DC cells), thereby generating an effective immune response.
  • Aluminum adjuvants have been successfully used since 1926. They can be administered safely and produce powerful immunity. Therefore, they are used in many human vaccines. However, it cannot stimulate the intracellular immune response, cannot induce Th1 immune response, and has the risk of potential local adverse reactions and hypersensitivity reactions. The quality is difficult to control and it is difficult to accurately evaluate the effect of adjuvants. Although aluminum adjuvants cannot effectively induce Th1 type responses, their application range is limited, but their safety and effectiveness in humans have been verified by time. Therefore, we believe that the construction of aluminum hydroxide as the core vaccine carrier will be very useful. potential.
  • One of the objectives of the present invention is to provide a vaccine carrier based on anionic polymers and derivatives thereof.
  • a series of anionic polymers and their derivatives are compounded with aluminum salts to form aluminum hydroxide nanoparticles, and different antigen components can be added during the preparation process to encapsulate the antigen.
  • the prepared vaccine can be efficiently taken up by antigen-presenting cells, delivered to lymph nodes and induce antigen-specific immune responses.
  • Aluminum hydroxide is an amphoteric compound, which forms a white precipitate that is insoluble in water at pH 6-10.
  • methods such as high-temperature calcination, hydrothermal reaction, and inverse microemulsion method are usually used to prepare nano-scale aluminum hydroxide.
  • the reaction conditions are severe and the process is complicated and cumbersome.
  • the nano-scale aluminum hydroxide is easy to agglomerate and disperse. Poor performance, poor stability, not suitable for clinical use.
  • the invention utilizes the characteristics that aluminum sulfate can generate aluminum hydroxide under alkaline conditions, and at the same time uses the positively charged and negatively charged characteristics of aluminum hydroxide and the negatively charged characteristics of the anionic polymer material.
  • the anionic polymer material is added to allow it to pass between the aluminum hydroxide and the aluminum hydroxide.
  • the electrostatic effect effectively restricts the aggregation and growth of aluminum hydroxide precipitation, thereby preparing nano-scale aluminum hydroxide with good stability and easy dispersion.
  • the anionic polymer material is the key to the generation of nano aluminum hydroxide.
  • One of the objectives of the present invention is to provide a vaccine carrier prepared based on an anionic polymer and its derivatives.
  • the aluminum salt is aluminum sulfate, and based on parts by weight, the anionic polymer material: aluminum sulfate is 0.06 to 4.8:0.16 -6.6.
  • anionic polymer in a broad sense in the art, refers to a polymeric material or polymer containing multiple anionic groups per molecule. It includes natural or endogenous, semi-synthetic derived, or fully synthetic polymers containing multiple anionic groups such as carboxyl, sulfate, sulfite, phosphate, phosphite and combinations thereof.
  • anionic polymer derivative includes "anion-derived polymer", which refers to a polymer that was not previously an anionic, but is converted into an anionic polymer by a suitable derivatization reactant, or is an anionic polymer itself and then derivatized The polymer still has anionic character.
  • derivatization reactions are carboxymethylation reaction, succinylation reaction, or maleylation reaction of carboxyl group, sulfation reaction of sulfate and sulfonate, sulfonation reaction, sulfination reaction, phosphate, phosphate The phosphating reaction, the phosphorylation reaction.
  • One of the objectives of the present invention is to provide a vaccine carrier prepared based on anionic polymers and derivatives thereof, characterized in that the natural or endogenous anionic polymer materials include: ⁇ -polyglutamic acid, mucopolysaccharides, and polymannan Uronic acid, polyguluronic acid, hyaluronic acid, chondroitin, heparin, keratan, alginic acid, dextran, xymann sulfate, fucoidan, fucogalactan, alginate , Agar, gellan gum, Indian gum, carrageenan, tragacanth, blue gum, xanthan gum, carrageenan; the semi-synthetic derivative anionic polymer material includes: heparin sulfate , Chondroitin sulfate, keratan sulfate, dextran sulfate, carboxymethyl cellulose, cross-linked caramel, carboxymethyl starch, carboxymethyl dextran, carboxymethyl
  • the anionic polymer can be derivatized and modified, but is not limited to PEG, carboxyl, sulfate, sulfite, phosphate, and phosphite modifications.
  • the anionic polymer can be a linear polymer, a cross-linked polymer, or a branched copolymer.
  • the anionic polymer may be a homopolymer or a copolymer.
  • the anionic polymer when it is a homopolymer, it contains a single type of repeating unit.
  • the anionic polymer is a copolymer, it contains two or more different repeating units.
  • the present invention selects three anionic polymer materials of ⁇ -polyglutamic acid, polyglutamic acid grafted polyethylene glycol and chondroitin sulfate.
  • the ⁇ -polyglutamic acid in the present invention is obtained by biological fermentation, with a molecular weight of 1,000 to 100,000 Daltons, and its structural formula is as follows
  • the polyglutamic acid is grafted with polyethylene glycol, and the length of the polyglutamic acid unit is 50-220; the length of the grafted polyethylene glycol unit is 500-1200, and the molecular weight of the polymer is 30,000-70,000 Daltons.
  • chondroitin sulfate The structure of chondroitin sulfate in the present invention is as follows
  • One of the objectives of the present invention is to provide a vaccine carrier prepared based on an anionic polymer and its derivatives, which is characterized in that the aluminum salt nanoparticles of the anionic polymer and its derivatives form a vaccine with the antigen through direct adsorption.
  • One of the objectives of the present invention is to provide a vaccine carrier prepared based on anionic polymers and derivatives thereof, characterized in that the antigen is selected from: protein antigens: hepatitis A, hepatitis B or hepatitis C antigen, tetanus Toxin, human papilloma virus, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, herpes simplex virus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, respiratory tract Syncytial virus, West Nile virus, cytomegalovirus, malaria antigen, Streptococcus pneumoniae, Legionella pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, group A streptococcus antigen, or other recombinants Protein antigens; protein
  • One of the objectives of the present invention is to provide a vaccine carrier prepared based on anionic polymer and its derivatives, which is characterized in that while the antigen is encapsulated, the nanoparticle itself has a certain adjuvant effect, and different adjuvants can also be added to combine further Improve immune response.
  • Adjuvant selected from: antigen-related molecular pattern adjuvant: Toll-like receptor agonist: peptidoglycan, lipoteichoic acid, MPLA, imiquimod, requimod, CpG-ODN, bacterial flagellin, Poly I:C; RIG-I-like receptor agonist: 3pRNA, short double-stranded RNA; NOD-like receptor agonist: muramyl dipeptide (MDP), N-acetylglucosamine; C-type lectin receptor : ⁇ -glucan, trehalose diborate; STING agonist: cGAMP; bacterial toxins and derivatives: cholera toxin (CT), Escherichia coli heat labile enterotoxin (LT), cholera toxin B subunit; Saponins: QS21, tomato glycoside, Quil-A; cytokines: GM-CSF, IL-2, IL-12, IL-6, IFN- ⁇ , Flt-3,
  • One of the objectives of the present invention is to provide a vaccine carrier prepared based on an anionic polymer and its derivatives, which is characterized by including the following steps:
  • step (1) the amount of the anionic polymer and its derivatives described in step (1) is 0.12-2.4mg
  • step (2) the amount of aluminum sulfate described in step (2) is 0.33-3.3 mg.
  • the diameter of the nanoparticles prepared according to the above steps is 30-200 nm, as shown in the example results in Table 1.
  • the potential is +10 ⁇ -30mv, and the vortex time of step (3) is 5-60s.
  • the power of the ultrasound in step (3) is 50-300W, and the time is 1-30min.
  • the preparation method of the present invention is simple and rapid, the conditions are mild, no organic reagents are added, it will not cause the denaturation of the protein, and can effectively maintain the conformation and activity of the protein.
  • the present invention selects OVA and HBsAg as model antigens, and experiments in vivo and in vitro prove that the vaccine carrier can induce antigen-specific humoral and cellular immunity.
  • the prepared vaccine vector can be efficiently taken up by DC2.4 and Raw264.7 antigen-presenting cells.
  • the aluminum content of the prepared vaccine carrier is 0.105mg/ml
  • it is compatible with the commercial hepatitis B vaccine (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) with aluminum content of 0.35-0.62mg/ml).
  • the produced antibody IgG and IgG2a subtype are better.
  • the vaccine carrier of the present invention uses anionic materials to modify aluminum hydroxide, has higher stability, is not easy to aggregate, has good dispersibility, and is more suitable for clinical use.
  • the vaccine carrier of the present invention has a particle size of 30-200 nm, which meets the requirements of lymph node delivery, can effectively target the lymph nodes, and realize the targeted delivery of the vaccine.
  • the vaccine carrier of the present invention can be efficiently taken up by the antigen-presenting cells, which is beneficial to the occurrence of the next immune response.
  • the vaccine carrier of the present invention can contain different kinds of antigens, and the nanoparticle itself has an adjuvant effect, which can help induce an antigen-specific immune response, thereby producing a stronger immune effect, and the two have a synergistic effect.
  • the vaccine carrier of the invention has a simple preparation method, no organic solvent is added, good repeatability and high stability.
  • the vaccine carrier of the present invention has low aluminum content, which is far lower than commercial aluminum gel ( 2% (InvivoGene, USA), which can reduce the danger of metal ion accumulation and local side effects, has low toxicity, and improves the adaptability of patients; at the same time, the aluminum content is much lower than the commercial aluminum gel adsorption vaccine (recombinant hepatitis B In the case of the vaccine (Saccharomyces cerevisiae) Shenzhen Kangtai), the induced immune response is equivalent to or stronger than that of the commercial aluminum gel, which has a better immune effect.
  • commercial aluminum gel adsorption vaccine recombinant hepatitis B In the case of the vaccine (Saccharomyces cerevisiae) Shenzhen Kangtai
  • the present invention has the following advantages:
  • Aluminum hydroxide nanoparticles formed by the composite of anionic polymer materials and aluminum salts are used as vaccine carriers, which expands the range of materials that can be used for encapsulating aluminum salts and improves the universality.
  • Aluminum hydroxide nanoparticles formed by the composite of anionic polymer materials and aluminum salts are used as vaccine carriers, and stable nanoparticles can be formed when materials without PEG modification are used.
  • the nanoparticles have good dispersibility and high stability.
  • the anionic polymer material used in the present invention has good biocompatibility, low toxicity and high safety.
  • chondroitin sulfate which is a kind of mucopolysaccharide with good biocompatibility, is widely present in cartilage tissues of humans and animals. It is used as a food for the treatment of osteoarthritis; at the same time, the FDA approved it as a skin Alternatives, chondroitin sulfate tablets and chondroitin sulfate injections are also on the market in my country, which are much safer than synthetic polymer materials and are cheap and easy to obtain.
  • the method of the present invention is simple, and the method of ultrasound and syringe pump can be used for large-scale production, which is beneficial to industrial transformation and has broad application and market prospects.
  • Figure 1 is a graph of the particle size of ⁇ -polyglutamic acid-nanoparticles.
  • Figure 2 shows the particle size of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles.
  • Figure 3 is a diagram showing the particle size of chondroitin sulfate-nanoparticles.
  • Figure 4 is an electron micrograph of ⁇ -polyglutamic acid-nanoparticles.
  • Figure 5 is an electron micrograph of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles.
  • Figure 6 is an electron micrograph of chondroitin sulfate-nanoparticles.
  • Figure 7 shows the uptake of antigen and antigen-carrying gamma-polyglutamic acid-nanoparticles (PGA) by DC2.4 and Raw264.7 cells.
  • PGA gamma-polyglutamic acid-nanoparticles
  • Figure 8 shows the uptake of antigens and antigen-carrying chondroitin sulfate-nanoparticles (ASN) by DC2.4 and Raw264.7 cells.
  • Figure 9 shows the retention of ⁇ -polyglutamic acid-nanoparticles (PGA) in lymph nodes.
  • Figure 10 shows the CTL results of ⁇ -polyglutamic acid-nanoparticles (PGA).
  • Figure 11 shows immune antibodies against ⁇ -polyglutamic acid-nanoparticles (PGA).
  • Figure 12 shows the immune antibody of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles (PGN).
  • Figure 13 shows immune antibodies against chondroitin sulfate-nanoparticles (ASN).
  • Preparation of aluminum hydroxide nanoparticles based on ⁇ -polyglutamic acid material add 125ul 1mg/ml ⁇ -polyglutamic acid to 345ul 80mmol/L Hepes buffer with a pH of 8, mix well, and draw 550ul 1.76mmol/ The aluminum sulfate solution of L is added to the above-mentioned solution, sonicated for 5 minutes, and the power is 120w, and it is obtained.
  • Preparation of aluminum hydroxide nanoparticles based on ⁇ -polyglutamic acid material add 120ul 8mg/ml ⁇ -polyglutamic acid to 360ul 100mmol/L Hepes buffer with a pH of 8, mix well, and draw 555ul 17.56mmol/ The aluminum sulfate solution of L is added to the above-mentioned solution, sonicated for 10 minutes, and the power is 150w, and it is obtained.
  • Preparation of aluminum hydroxide nanoparticles-OVA based on ⁇ -polyglutamic acid material Add 130ul 20mg/ml ⁇ -polyglutamic acid to 370ul 100mmol/LPH of 8 Hepes buffer, mix well, 450ul absorb 2.16mmol /L aluminum sulfate solution and 100ul 4mg/ml OVA solution are mixed uniformly, add to the above solution, ultrasonic 8min, power 100w, and it is obtained.
  • Preparation of aluminum hydroxide nanoparticles-OVA based on ⁇ -polyglutamic acid material add 140ul 7.5mg/ml ⁇ -polyglutamic acid to 400ul 70mmol/LPH of 8 Hepes buffer, mix well, and draw 460ul 1.4 Mix the mmol/L aluminum sulfate solution and 100ul 1mg/ml OVA solution uniformly, add them to the above solution, sonicate for 6min, and the power is 150w, and it will be obtained.
  • Preparation of aluminum hydroxide nanoparticles-OVA-CpG based on the ⁇ -polyglutamic acid material Add 110ul 10mg/ml ⁇ -polyglutamic acid to 380ul 80mmol/L Hepes buffer with a pH of 8, mix well, and absorb Mix 500ul 2mmol/L aluminum sulfate solution, 100ul 2mg/ml OVA solution and 10ul 2mg/ml CpG solution, add to the above solution, sonicate for 5min, power 120w, and get it.
  • Preparation of aluminum hydroxide nanoparticles-OVA-CpG based on ⁇ -polyglutamic acid material add 100ul 5mg/ml ⁇ -polyglutamic acid to 400ul 50mmol/L Hepes buffer with a pH of 8, mix well, and absorb Mix 560ul 1.5mmol/L aluminum sulfate solution, 50ul 4mg/ml OVA solution and 10ul 500ug/ml CpG solution evenly, add to the above solution, sonicate for 15min, power 100w, and get it.
  • Preparation of aluminum hydroxide nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 125ul 2.5mg/ml polyglutamic acid grafted polyethylene glycol to 340ul 100mmol/L Hepes buffer with a pH of 8 Mix the materials evenly, add 560ul 1.6mmol/L aluminum sulfate solution to the above mixed solution, and vortex for 30s to get.
  • Preparation of aluminum hydroxide nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 110ul 7.5mg/ml polyglutamic acid grafted polyethylene glycol to 360ul 80mmol/L Hepes buffer with a pH of 8 Mix the materials evenly, add 555ul 2mmol/L aluminum sulfate solution to the above mixed solution, and vortex for 30s to get.
  • Preparation of aluminum hydroxide nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 100ul 10mg/ml polyglutamic acid grafted polyethylene glycol material to 350ul 100mmol/L Hepes buffer with a pH of 8 , Mix well, add 500ul 5.5mmol/L aluminum sulfate solution to the above mixed solution, vortex for 30s to get.
  • Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 110ul 10mg/ml of polyglutamic acid grafted polyethylene to 345ul 100mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 300ul 6.6mmol/L aluminum sulfate solution and 220ul 50ug/ml HBsAg solution and mix well, add to the above mixed solution, vortex for 30s to get.
  • Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 125ul 10mg/ml of polyglutamic acid grafted polyethylene to 400ul 90mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 290ul 9mmol/L aluminum sulfate solution and 200ul 400ug/ml HBsAg solution and mix evenly, add to the above mixed solution, vortex for 30s to get.
  • Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 125ul 12mg/ml of polyglutamic acid grafted polyethylene to 400ul 100mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 250ul 10mmol/L aluminum sulfate solution and 250ul 20ug/ml HBsAg solution and mix evenly, add to the above mixed solution, vortex for 30s to get.
  • Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material add 120ul 10mg/ml of polyglutamic acid grafted polyethylene to 380ul 90mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 280ul 8mmol/L aluminum sulfate solution and 240ul 10ug/ml HBsAg solution and mix evenly, add to the above mixed solution, vortex for 30s to get.
  • Preparation of aluminum hydroxide nanoparticles based on chondroitin sulfate add 220ul 10mg/ml chondroitin sulfate material to 340ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, suck 400ul 10mmol/L aluminum sulfate solution, add In the above mixed solution, vortex for 30 seconds.
  • Preparation of aluminum hydroxide nanoparticles based on chondroitin sulfate add 200ul 10mg/ml chondroitin sulfate material to 400ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, suck 380ul 10mmol/L aluminum sulfate solution, add In the above mixed solution, vortex for 30 seconds.
  • Preparation of aluminum hydroxide-OVA nanoparticles based on chondroitin sulfate Add 300ul 10mg/ml chondroitin sulfate material to 400ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, and draw 340ul 10mmol/L aluminum sulfate solution Mix well with 60ul 1mg/ml OVA solution, add to the above mixture, vortex for 30s to get.
  • Preparation of aluminum hydroxide-OVA-CpG nanoparticles based on chondroitin sulfate add 280ul 10mg/ml chondroitin sulfate material to 360ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, and absorb 350ul 10mmol/L sulfuric acid Mix the aluminum solution with 70ul 0.85mg/ml OVA solution and 20ul 2mg/ml CpG solution evenly, add to the above mixture, and vortex for 30s to get.
  • Preparation of aluminum hydroxide-OVA nanoparticles based on chondroitin sulfate add 10ml 10mg/ml chondroitin sulfate material and 3ml 1mg/ml OVA solution to 20ml 100mmol/L Hepes buffer with a pH of 7.8, mix well and add 1 No. 2 syringe, 33ml 6.06mmol/L aluminum sulfate solution is added to No. 2 syringe, and the two syringes are simultaneously passed through the micro-syringe pump at a speed of 20ml/min through the special-shaped three-channel microfluidic device, and the mixed liquid is collected, which is the nanoparticle.
  • Nanoparticle size measurement use Zetasizer Nano ZS90 laser particle size analyzer to measure the ⁇ -polyglutamic acid-nanoparticles, polyglutamic acid grafted polyethylene glycol-HBsAg, and chondroitin sulfate-nanoparticles of Examples 1-19 Take 1ml of the nanoparticle solution of Example 1-19, put the sample into the sample cell, and set the measurement temperature to 25°C. The results are shown in Table 1. The results show that the nanoparticle size is about 100nm. , PDI meets the requirements and the distribution is uniform.
  • Measurement of the particle size of ⁇ -polyglutamic acid-nanoparticles The particle size distribution of the ⁇ -polyglutamic acid-nanoparticles of Example 1 was measured using a Zetasizer Nano ZS90 laser particle size analyzer. Take 1ml of the nanoparticle solution, put the sample into the sample cell, and set the measurement temperature to 25°C. The result is shown in Figure 1. The size of the nanoparticles is about 100nm, and the distribution is uniform.
  • Determination of the particle size of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles The particle size distribution of the polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles of Example 11 was measured using a Zetasizer Nano ZS90 laser particle size analyzer . Take 1ml of the nanoparticle solution, put the sample into the sample cell, and set the measurement temperature to 25°C. The result is shown in Figure 2. The size of the nanoparticles is about 100nm, and the distribution is uniform.
  • Determination of the particle size of chondroitin sulfate-nanoparticles The particle size distribution of the chondroitin sulfate-nanoparticles of Example 16 was measured using a Zetasizer Nano ZS90 laser particle size analyzer. Take 1ml of the nanoparticle solution, put the sample into the sample cell, and set the measurement temperature to 25°C. The result is shown in Figure 3. The size of the nanoparticles is about 100nm, and the distribution is uniform.
  • ⁇ -Polyglutamic Acid-Nanoparticles Photo Transmission Electron Microscope Place the ⁇ -Polyglutamic Acid-Nanoparticles sample of Example 1 on a copper mesh, let stand for 5 minutes, then stain with phosphotungstic acid for 1 minute, and then suck it away with filter paper Excess dye solution on the copper mesh, dry the sample at room temperature, and observe the sample under a transmission electron microscope at 200kv. The results are shown in Fig. 4, and it can be seen from the experimental results that the nanoparticles are all round particles with a particle size below 100 nm.
  • Chondroitin sulfate-nanoparticles according to transmission electron microscopy the chondroitin sulfate-nanoparticles sample of Example 16 was placed on a copper mesh, allowed to stand for 5 min, and then stained with phosphotungstic acid for 1 min, and then filter paper was used to absorb the excess dye solution on the copper mesh , Dry the sample at room temperature, and observe the sample under a transmission electron microscope under 200kv. The results are shown in Fig. 6, and it can be seen from the experimental results that the nanoparticles are all round particles with a particle size below 100 nm.
  • Uptake of ⁇ -polyglutamic acid-nanoparticles in DC2.4 and Raw264.7 cells In a twelve-well plate, plant 1 ⁇ 10 6 DC2.4 or Raw264.7 cells in each well, and put them in Incubate for 4-6 hours, after the cells adhere to the wall, add 50 ⁇ l of FITC-labeled OVA or the ⁇ -polyglutamic acid-nanoparticle of Example 1 prepared with FITC-labeled OVA to each well.
  • mice C57BL/6 mice were injected with 25 ⁇ l FITC-labeled OVA into the ⁇ -polyglutamic acid-nanoparticle solution of Example 1 and administered for 4 hours Twenty hours later, the mice were sacrificed by cervical dislocation, and the lymph nodes at the popliteal curve were separated.
  • the nanoparticles reached the lymph nodes within 4 hours, and about 5% of FITC-positive cells were detected, indicating that the ⁇ -polyglutamic acid-nanoparticles have lymph node targeting ability. At 20 hours, about 1% of FITC-positive cells could still be detected in the lymph nodes, indicating that the ⁇ -polyglutamic acid-nanoparticles exhibited a certain retention ability in the lymph nodes.
  • the flow cytometry antibody stained the characteristic surface molecule CD11c of DC cells, distinguishing DC cells from the lymph node cell population.
  • the FITC+CD11c+ double positive signal is the DC cells that have taken up ⁇ -polyglutamic acid nanoparticles, as shown in Figure 9b As shown, most of the nanoparticles in the lymph nodes are taken up by DC cells, which also provides favorable conditions for the occurrence of the next immune response.
  • Cytotoxic T lymphocyte (CTL) test and immune antibody detection of ⁇ -polyglutamic acid-nanoparticles On day 0 and day 7, mice were injected with 25ul of ⁇ -polyglutamic acid from Example 5 on the soles of their feet. Nanoparticles (each 5ugOVA, 0.5ug CpG), on the 14th day by CFSE staining method to detect in vivo CTL response. The results are shown in Figure 10, ⁇ -polyglutamic acid-nanoparticles can produce a strong antigen-specific cellular immune response, and its CTL is significantly higher than that of the free OVA+CpG group, with a significant difference (P ⁇ 0.001).
  • mice were injected with 25ul of the ⁇ -polyglutamic acid-nanoparticles of Example 5 in the sole of the foot, (each 5ugOVA, 0.5ug CpG ), blood was taken from the orbit on the 21st day, and OVA-specific antibodies in the serum were detected.
  • the results are shown in Figure 11, where Figure 11a, Figure 11b and Figure 11c are the antibody detection results of IgG, IgG1 and IgG2a, respectively.
  • mice were injected with 25ul of the polyglutamic acid grafted polyethylene glycol of Example 11 in the soles of the mice.
  • the dose of HBsAg was 2ugHBsAg per mouse.
  • Blood was taken from the orbit at 35 days to detect HBsAg-specific antibodies in the serum.
  • polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles can produce a strong antigen-specific immune response, and the IgG level is significantly higher than that of the free antigen group (P ⁇ 0.0001) and the commercially available vaccine group ( P ⁇ 0.001), the IgG2a level was also significantly higher than the free antigen group (P ⁇ 0.0001) and the commercial vaccine group (P ⁇ 0.01); the IgG1 level was significantly higher than the free antigen group (P ⁇ 0.01), which was comparable to the commercial vaccine group .
  • Polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles can induce stronger antibody levels than free antigen and commercial aluminum gel adsorption vaccines, indicating that they have a certain adjuvant effect.
  • chondroitin sulfate-nanoparticles can produce a strong antigen-specific immune response, and the IgG level is significantly higher than that of the free OVA+CpG group, with a significant difference (P ⁇ 0.01), and the IgG1 and IgG2a levels are also significant Higher than the free OVA+CpG group (P ⁇ 0.05).

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Abstract

Provided in the present invention is a vaccine vector prepared on the basis of anionic polymers and derivatives thereof, and a preparation method therefor. Nanoparticles are compounded with an aluminum salt by means of a series of anionic polymers and derivatives thereof to form aluminum hydroxide. In the preparation process, different antigen components are added, and the antigens may be encapsulated. The prepared vaccine can effectively be absorbed by an antigen presenting cell and transmitted to a lymph node, and induce an antigen-specific immune response.

Description

一种基于阴离子聚合物及其衍生物制备的疫苗载体Vaccine carrier prepared based on anionic polymer and its derivatives 技术领域Technical field
本发明涉及医药技术领域，具体涉及一种基于阴离子聚合物及其衍生物材料的疫苗载体及其制备方法。The invention relates to the technical field of medicine, in particular to a vaccine carrier based on anionic polymer and its derivative materials and a preparation method thereof.
背景技术Background technique
疫苗接种是现代医学的伟大胜利之一，也是大多数疾病预防和根除的有效手段，用于控制多种感染性疾病甚至根除了天花。一般疫苗主要是减毒或灭活疫苗，免疫原性高但安全性低。而现在的研究主要集中于亚单位疫苗，因为其更纯净安全。但是相比减毒疫苗，安全性的提高伴随着免疫原性的降低。因此，佐剂成为疫苗中至关重要的组分，用以提升亚单位疫苗持久有效的免疫效应。使用疫苗递送载体，能够诱导有效的免疫响应和提供改善的稳定性,安全性和成本和有效性。在过去十年中，纳米级(<1000nm)材料，如病毒样颗粒、脂质体、ISCOMS、聚合物和不可降解的纳米球，纳米粒等，作为疫苗抗原的递送载体而受到人们的关注，这种载体既可以稳定疫苗抗原，避免抗原的降解，其本身也有一定的佐剂效果，可以提高帮助抗原实现其免疫反应。Vaccination is one of the great victories of modern medicine. It is also an effective means of preventing and eradicating most diseases. It is used to control a variety of infectious diseases and even eradicate smallpox. General vaccines are mainly attenuated or inactivated vaccines, which have high immunogenicity but low safety. The current research is mainly focused on subunit vaccines because they are more pure and safe. However, compared with attenuated vaccines, the increase in safety is accompanied by a decrease in immunogenicity. Therefore, adjuvants have become a vital component in vaccines to enhance the lasting and effective immune effect of subunit vaccines. The use of vaccine delivery vehicles can induce an effective immune response and provide improved stability, safety, and cost and effectiveness. In the past ten years, nano-scale (<1000nm) materials, such as virus-like particles, liposomes, ISCOMS, polymers and non-degradable nanospheres, nanoparticles, etc., have received attention as delivery vehicles for vaccine antigens. This kind of carrier can stabilize the vaccine antigen and avoid the degradation of the antigen. It also has a certain adjuvant effect, which can help the antigen to achieve its immune response.
疫苗递送载体的大小会影响其分布并最终影响抗原应答。当由肌肉注射或皮下注射途径，大小为20–100nm的粒子可穿过细胞外基质，直接进入淋巴管。而更大粒径的粒子一般被皮下的DC细胞摄取再迁移到淋巴结。后者的速度和递送抗原量远远低于前者。通过设计纳米粒的性质，可以使纳米粒直接靶向到淋巴结，被大量的免疫细胞(如DC细胞)所捕获，从而产生有效的免疫应答。The size of the vaccine delivery vehicle will affect its distribution and ultimately affect the antigen response. When injected intramuscularly or subcutaneously, particles with a size of 20-100nm can pass through the extracellular matrix and directly enter the lymphatic vessels. Larger particles are generally taken up by DC cells under the skin and then migrate to the lymph nodes. The speed of the latter and the amount of antigen delivered are much lower than the former. By designing the properties of the nanoparticles, the nanoparticles can be directly targeted to the lymph nodes and captured by a large number of immune cells (such as DC cells), thereby generating an effective immune response.
铝佐剂自从1926年被成功地使用，可安全地给药，产生强大的免疫，因而用于多种人用疫苗。然而它不能刺激细胞内免疫反应，不能诱导Th1型免疫反应，且存在潜在的局部不良反应和超敏反应的危险，质量难控制且对佐剂的效果很难做出准确的评价。虽然铝佐剂无法有效诱导Th1型应答的特点限制了其应用范围，但其用于人体的安全有效性是被时间验证了的，因此我们认为构建氢氧化铝为核心的疫苗载体将会非常有潜力。Aluminum adjuvants have been successfully used since 1926. They can be administered safely and produce powerful immunity. Therefore, they are used in many human vaccines. However, it cannot stimulate the intracellular immune response, cannot induce Th1 immune response, and has the risk of potential local adverse reactions and hypersensitivity reactions. The quality is difficult to control and it is difficult to accurately evaluate the effect of adjuvants. Although aluminum adjuvants cannot effectively induce Th1 type responses, their application range is limited, but their safety and effectiveness in humans have been verified by time. Therefore, we believe that the construction of aluminum hydroxide as the core vaccine carrier will be very useful. potential.
随着纳米技术的不断发展和进步，我们也将目光也投向了铝佐剂，将铝佐剂制备成纳米颗粒后，其粒径更小，比表面积急剧增大，具有表面反应，活性高、活性中心多、吸附能力强等特性，在相同铝含量的情况下，可吸附 更多的抗原。With the continuous development and progress of nanotechnology, we have also set our sights on aluminum adjuvants. After aluminum adjuvants are prepared into nanoparticles, their particle size is smaller, the specific surface area increases sharply, surface reactions, high activity, It has many active centers and strong adsorption capacity. With the same aluminum content, it can adsorb more antigens.
因此存在将氢氧化铝通过加入一定材料限制其聚集生长，从而制备成纳米级别颗粒用于疫苗递送的需要。Therefore, there is a need to restrict the aggregation and growth of aluminum hydroxide by adding certain materials, so as to prepare nano-scale particles for vaccine delivery.
发明内容Summary of the invention
本发明的目的之一是提供一种基于阴离子聚合物及其衍生物制备的疫苗载体。本发明通过一系列阴离子聚合物及其衍生物与铝盐复合形成氢氧化铝纳米粒，在制备的过程中加入不同的抗原成分可将抗原包载。制备的疫苗可以高效地被抗原呈递细胞摄取，传递到淋巴结并诱导抗原特异性的免疫反应。One of the objectives of the present invention is to provide a vaccine carrier based on anionic polymers and derivatives thereof. In the present invention, a series of anionic polymers and their derivatives are compounded with aluminum salts to form aluminum hydroxide nanoparticles, and different antigen components can be added during the preparation process to encapsulate the antigen. The prepared vaccine can be efficiently taken up by antigen-presenting cells, delivered to lymph nodes and induce antigen-specific immune responses.
氢氧化铝是一种两性化合物，其在pH6～10之间生成不溶于水的白色沉淀。现有技术中，通常使用高温煅烧、水热反应、反相微乳法等方法制备纳米级氢氧化铝，其反应条件剧烈，而且过程复杂繁琐，制得纳米级别的氢氧化铝易团聚，分散性能差，稳定性差，不适合于临床使用。本发明利用硫酸铝能在碱性条件生成氢氧化铝的特点，同时利用氢氧化铝带正电荷和阴离子聚合物材料带负电的特性，加入阴离子聚合物材料，使其与氢氧化铝之间通过静电作用，有效地限制氢氧化铝沉淀的聚集生长，从而制备稳定性好、易于分散的纳米级的氢氧化铝。在此制备过程中，阴离子聚合物材料是纳米氢氧化铝生成的关键。Aluminum hydroxide is an amphoteric compound, which forms a white precipitate that is insoluble in water at pH 6-10. In the prior art, methods such as high-temperature calcination, hydrothermal reaction, and inverse microemulsion method are usually used to prepare nano-scale aluminum hydroxide. The reaction conditions are severe and the process is complicated and cumbersome. The nano-scale aluminum hydroxide is easy to agglomerate and disperse. Poor performance, poor stability, not suitable for clinical use. The invention utilizes the characteristics that aluminum sulfate can generate aluminum hydroxide under alkaline conditions, and at the same time uses the positively charged and negatively charged characteristics of aluminum hydroxide and the negatively charged characteristics of the anionic polymer material. The anionic polymer material is added to allow it to pass between the aluminum hydroxide and the aluminum hydroxide. The electrostatic effect effectively restricts the aggregation and growth of aluminum hydroxide precipitation, thereby preparing nano-scale aluminum hydroxide with good stability and easy dispersion. In this preparation process, the anionic polymer material is the key to the generation of nano aluminum hydroxide.
本发明的目的之一是提供一种基于阴离子聚合物及其衍生物制备的疫苗载体，所述铝盐为硫酸铝，其中基于重量份计，阴离子聚合物材料：硫酸铝为0.06～4.8:0.16-6.6。One of the objectives of the present invention is to provide a vaccine carrier prepared based on an anionic polymer and its derivatives. The aluminum salt is aluminum sulfate, and based on parts by weight, the anionic polymer material: aluminum sulfate is 0.06 to 4.8:0.16 -6.6.
术语“阴离子聚合物”,按本领域广义的理解,指每个分子包含多个阴离子基团的聚合材料或聚合物。它包括含有多个阴离子基团如羧基、硫酸根、亚硫酸根、磷酸根、亚磷酸根及其组合的天然或者内源性，半合成衍生，或者全合成的聚合物。The term "anionic polymer", in a broad sense in the art, refers to a polymeric material or polymer containing multiple anionic groups per molecule. It includes natural or endogenous, semi-synthetic derived, or fully synthetic polymers containing multiple anionic groups such as carboxyl, sulfate, sulfite, phosphate, phosphite and combinations thereof.
术语“阴离子聚合物衍生物”包括“阴离子衍生而来的聚合物”,指以前不是阴离子聚合物,由合适的衍生化反应物转变为阴离子聚合物，或者本身是阴离子聚合物，然后进行了衍生化仍然具有阴离子特性的聚合物。衍生反应的例子有羧基的羧甲基化反应、琥珀酰化反应、或马来酰化反应，硫酸盐，磺酸盐的硫酸化反应，磺化反应，亚磺化反应，磷酸盐，磷酸盐的磷化反应，磷酰化反应。The term "anionic polymer derivative" includes "anion-derived polymer", which refers to a polymer that was not previously an anionic, but is converted into an anionic polymer by a suitable derivatization reactant, or is an anionic polymer itself and then derivatized The polymer still has anionic character. Examples of derivatization reactions are carboxymethylation reaction, succinylation reaction, or maleylation reaction of carboxyl group, sulfation reaction of sulfate and sulfonate, sulfonation reaction, sulfination reaction, phosphate, phosphate The phosphating reaction, the phosphorylation reaction.
本发明的目的之一提供一种基于阴离子聚合物及其衍生物制备的疫苗载体，其特征在于所述天然或者内源性阴离子聚合物材料包括：γ-聚谷氨酸、粘多糖，聚甘露糖醛酸，聚古罗糖醛酸，透明质酸，软骨素，肝素、角质素、海藻酸、葡聚糖、硫酸木甘露聚糖、岩藻多糖、岩藻半乳聚糖、海藻酸盐、琼脂、吉兰糖胶、印度树胶、卡拉亚胶、黄耆胶、兰胶、黄原胶、角叉菜胶的一种或多种；所述半合成衍生阴离子聚合物材料包括：硫酸肝素、硫酸软骨素、硫酸角质素、硫酸葡聚糖、羧甲基纤维素、交联焦糖、羧甲基淀粉、羧甲基葡聚糖、羧甲基壳聚糖、透明质酸衍生物、硫酸鼠李多糖、硫酸纤维素、硫酸凝胶多糖和磷酸壳聚糖的一种或多种；所述全合成阴离子聚合物材料包括：聚阴离子多肽、聚丙烯酸、聚甲基丙烯酸、聚谷氨酸、聚天冬氨酸、聚天冬氨酸接枝聚乙二醇、聚谷氨酸接枝聚乙二醇、聚卡波菲尔、羧乙烯聚合物、马来酸酐共聚物、硫醇化聚丙烯酸酯的一种或多种。One of the objectives of the present invention is to provide a vaccine carrier prepared based on anionic polymers and derivatives thereof, characterized in that the natural or endogenous anionic polymer materials include: γ-polyglutamic acid, mucopolysaccharides, and polymannan Uronic acid, polyguluronic acid, hyaluronic acid, chondroitin, heparin, keratan, alginic acid, dextran, xymann sulfate, fucoidan, fucogalactan, alginate , Agar, gellan gum, Indian gum, carrageenan, tragacanth, blue gum, xanthan gum, carrageenan; the semi-synthetic derivative anionic polymer material includes: heparin sulfate , Chondroitin sulfate, keratan sulfate, dextran sulfate, carboxymethyl cellulose, cross-linked caramel, carboxymethyl starch, carboxymethyl dextran, carboxymethyl chitosan, hyaluronic acid derivatives, Rhamn sulfate, cellulose sulfate, curdlan sulfate, and chitosan phosphate; the fully synthetic anionic polymer material includes: polyanionic polypeptide, polyacrylic acid, polymethacrylic acid, polyglutamine Acid, polyaspartic acid, polyaspartic acid grafted polyethylene glycol, polyglutamic acid grafted polyethylene glycol, polycarbophil, carboxyvinyl polymer, maleic anhydride copolymer, thiolated One or more of polyacrylate.
所述的阴离子聚合物可以进行衍生化修饰，但不限于PEG，羧基，硫酸根、亚硫酸根、磷酸根、亚磷酸根修饰。The anionic polymer can be derivatized and modified, but is not limited to PEG, carboxyl, sulfate, sulfite, phosphate, and phosphite modifications.
所述的阴离子聚合物可以是线性聚合物，交联聚合物，或支化共聚物。The anionic polymer can be a linear polymer, a cross-linked polymer, or a branched copolymer.
所述的阴离子聚合物可以是均聚物或者共聚物，当阴离子聚合物是均聚物时，包含单一类型的重复单元。当阴离子聚合物是共聚物时，包含两个或者更多不同的重复单元。The anionic polymer may be a homopolymer or a copolymer. When the anionic polymer is a homopolymer, it contains a single type of repeating unit. When the anionic polymer is a copolymer, it contains two or more different repeating units.
作为优选的实施方案，本发明选择γ-聚谷氨酸，聚谷氨酸接枝聚乙二醇和硫酸软骨素三种阴离子聚合物材料。As a preferred embodiment, the present invention selects three anionic polymer materials of γ-polyglutamic acid, polyglutamic acid grafted polyethylene glycol and chondroitin sulfate.
本发明中的γ-聚谷氨酸为生物发酵得到，分子量为1000-100000道尔顿其结构式如下图The γ-polyglutamic acid in the present invention is obtained by biological fermentation, with a molecular weight of 1,000 to 100,000 Daltons, and its structural formula is as follows
Figure PCTCN2020136150-appb-000001
Figure PCTCN2020136150-appb-000001
本发明中的聚谷氨酸接枝聚乙二醇，聚谷氨酸单元长度50-220；接枝的聚乙二醇单元长度500-1200，聚合物的分子量在30000-70000道尔顿。In the present invention, the polyglutamic acid is grafted with polyethylene glycol, and the length of the polyglutamic acid unit is 50-220; the length of the grafted polyethylene glycol unit is 500-1200, and the molecular weight of the polymer is 30,000-70,000 Daltons.
其结构式如下图Its structural formula is as follows
Figure PCTCN2020136150-appb-000002
Figure PCTCN2020136150-appb-000002
本发明中的硫酸软骨素结构如下图The structure of chondroitin sulfate in the present invention is as follows
Figure PCTCN2020136150-appb-000003
Figure PCTCN2020136150-appb-000003
本发明的目的之一提供一种基于阴离子聚合物及其衍生物制备的疫苗载体，其特征在于阴离子聚合物及其衍生物的铝盐纳米粒通过直接吸附作用与抗原形成疫苗。One of the objectives of the present invention is to provide a vaccine carrier prepared based on an anionic polymer and its derivatives, which is characterized in that the aluminum salt nanoparticles of the anionic polymer and its derivatives form a vaccine with the antigen through direct adsorption.
本发明的目的之一提供一种基于阴离子聚合物及其衍生物制备的疫苗载体，其特征在于所述抗原选自：蛋白质抗原：甲型肝炎，乙型肝炎或丙型肝炎抗原、破伤风类毒素、人乳头瘤病毒、白喉毒素、霍乱毒素、百日咳毒素、乙型脑炎病毒、流感病毒、结核、单纯疱疹病毒、麻疹病毒、风疹病毒、腮腺炎病毒、埃博拉病毒、狂犬病毒、呼吸道合胞病毒、西尼罗病毒、巨细胞病毒、疟疾抗原、肺炎链球菌、侵肺军团菌、脑膜炎奈瑟菌、铜绿假单胞菌、霍乱弧菌、A组链球菌抗原、或者其他重组蛋白抗原；免疫原性较弱的蛋白抗原，包括：牛血清白蛋白、溶菌酶、转铁蛋白、胰岛素、乳白蛋白、肌白蛋白、豆白蛋白、麦白蛋白、肌红蛋白、胶原蛋白、纤层蛋白；多肽抗原，包括：TRP2、HGP100、p15E、E6、E7、SIINFEKL、乙肝抗原表位肽 S28-39、或者其他合成的多肽抗原及含有几种多肽序列的长多肽抗原；病毒或细菌裂解液抗原、病毒或细菌外膜囊泡抗原、肿瘤细胞裂解液抗原，肿瘤细胞膜囊泡抗原、肿瘤细胞外泌体抗原或者肿瘤模式抗原鸡卵清白蛋白(OVA)的一种或多种。One of the objectives of the present invention is to provide a vaccine carrier prepared based on anionic polymers and derivatives thereof, characterized in that the antigen is selected from: protein antigens: hepatitis A, hepatitis B or hepatitis C antigen, tetanus Toxin, human papilloma virus, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, herpes simplex virus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, respiratory tract Syncytial virus, West Nile virus, cytomegalovirus, malaria antigen, Streptococcus pneumoniae, Legionella pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, group A streptococcus antigen, or other recombinants Protein antigens; protein antigens with weak immunogenicity, including: bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myosin, soy albumin, wheat albumin, myoglobin, collagen, Laminin; polypeptide antigens, including: TRP2, HGP100, p15E, E6, E7, SIINFEKL, hepatitis B epitope peptide S28-39, or other synthetic polypeptide antigens and long polypeptide antigens containing several polypeptide sequences; viruses or bacteria One or more of lysate antigen, virus or bacterial outer membrane vesicle antigen, tumor cell lysate antigen, tumor cell membrane vesicle antigen, tumor cell exosomal antigen, or tumor model antigen chicken ovalbumin (OVA).
本发明的目的之一提供一种基于阴离子聚合物及其衍生物制备的疫苗载体，其特征在于包载抗原的同时，纳米粒本身具有一定的佐剂作用，也可加入不同的佐剂联合进一步提高免疫反应。佐剂选自：抗原相关分子模式类佐剂：Toll样受体激动剂：肽聚糖、脂磷壁酸、MPLA、咪喹莫特、瑞喹莫特、CpG-ODN、细菌鞭毛蛋白、Poly I:C；RIG-I样受体激动剂：3pRNA、短的双链RNA；NOD样受体激动剂：胞壁酰二肽(MDP)、N一乙酰葡萄糖胺；C-型凝集素受体：β-葡聚糖、海藻糖二硼酸盐；STING激动剂：cGAMP；细菌毒素及其衍生物：霍乱毒素(CT)、大肠杆菌不耐热肠毒素(LT)、霍乱毒素B亚单位；皂苷类：QS21、番茄苷、Quil-A；细胞因子：GM-CSF、IL-2、IL-12、IL-6、IFN-γ、Flt-3、淋巴细胞趋化因子；其他佐剂：热激蛋白、GTP-GDP、氟化钠、烷基聚丙烯酯多聚体、二甲基双十八烷基季胺溴化物(DDA)的一种或多种。One of the objectives of the present invention is to provide a vaccine carrier prepared based on anionic polymer and its derivatives, which is characterized in that while the antigen is encapsulated, the nanoparticle itself has a certain adjuvant effect, and different adjuvants can also be added to combine further Improve immune response. Adjuvant selected from: antigen-related molecular pattern adjuvant: Toll-like receptor agonist: peptidoglycan, lipoteichoic acid, MPLA, imiquimod, requimod, CpG-ODN, bacterial flagellin, Poly I:C; RIG-I-like receptor agonist: 3pRNA, short double-stranded RNA; NOD-like receptor agonist: muramyl dipeptide (MDP), N-acetylglucosamine; C-type lectin receptor : Β-glucan, trehalose diborate; STING agonist: cGAMP; bacterial toxins and derivatives: cholera toxin (CT), Escherichia coli heat labile enterotoxin (LT), cholera toxin B subunit; Saponins: QS21, tomato glycoside, Quil-A; cytokines: GM-CSF, IL-2, IL-12, IL-6, IFN-γ, Flt-3, lymphocyte chemotactic factor; other adjuvants: heat One or more of kinin, GTP-GDP, sodium fluoride, alkyl polyacrylate polymer, dimethyl dioctadecyl quaternary ammonium bromide (DDA).
本发明的目的之一提供一种基于阴离子聚合物及其衍生物制备的疫苗载体，其特征在于包括以下步骤：One of the objectives of the present invention is to provide a vaccine carrier prepared based on an anionic polymer and its derivatives, which is characterized by including the following steps:
(1)向Hepes缓冲液中加入阴离子聚合物或其衍生物材料，混合均匀(1) Add anionic polymer or its derivative materials to Hepes buffer and mix well
(2)将硫酸铝和含或不含抗原溶液混合均匀，加入上述溶液中(2) Mix aluminum sulfate and the solution with or without antigen, and add it to the above solution
(3)涡旋、超声或者通过微量注射泵进行混合。(3) Mix by vortexing, ultrasonic or micro-syringe pump.
作为优选的实施方案，以最终纳米粒体积1ml计算：As a preferred embodiment, it is calculated based on the final nanoparticle volume of 1ml:
其中步骤(1)所述的阴离子聚合物及其衍生物的量为0.12-2.4mg，Wherein the amount of the anionic polymer and its derivatives described in step (1) is 0.12-2.4mg,
其中步骤(1)所述的Hepes缓冲液的量为18.5-185umol，Wherein the amount of Hepes buffer described in step (1) is 18.5-185umol,
其中步骤(2)所述的硫酸铝的量为0.33-3.3mg。Wherein, the amount of aluminum sulfate described in step (2) is 0.33-3.3 mg.
根据本发明按上述步骤制备出的纳米粒粒径为30～200nm，如表一的实施例结果所示。电位为+10～-30mv，其中步骤(3)的涡旋时间为5-60s。According to the present invention, the diameter of the nanoparticles prepared according to the above steps is 30-200 nm, as shown in the example results in Table 1. The potential is +10～-30mv, and the vortex time of step (3) is 5-60s.
其中步骤(3)的超声的功率为50-300W,时间为1-30min。The power of the ultrasound in step (3) is 50-300W, and the time is 1-30min.
本发明所述的制备方法简单迅速，条件温和，无有机试剂的加入，不会 造成蛋白的变性，可以有效地保持蛋白的构象和活性。The preparation method of the present invention is simple and rapid, the conditions are mild, no organic reagents are added, it will not cause the denaturation of the protein, and can effectively maintain the conformation and activity of the protein.
本发明选择OVA和HBsAg作为模型抗原，通过体内外实验证明，该疫苗载体可诱导抗原特异性的体液和细胞免疫。The present invention selects OVA and HBsAg as model antigens, and experiments in vivo and in vitro prove that the vaccine carrier can induce antigen-specific humoral and cellular immunity.
作为本发明优选的实施方案之一，制备的疫苗载体可被DC2.4和Raw264.7两种抗原提呈细胞高效地摄取。As one of the preferred embodiments of the present invention, the prepared vaccine vector can be efficiently taken up by DC2.4 and Raw264.7 antigen-presenting cells.
作为本发明优选的实施方案之一，制备的疫苗载体的铝含量为0.105mg/ml时，与铝含量为0.35-0.62mg/ml的商品化乙肝疫苗(重组乙型肝炎疫苗(酿酒酵母)深圳康泰)相比，产生的抗体IgG以及IgG2a亚型更优。As one of the preferred embodiments of the present invention, when the aluminum content of the prepared vaccine carrier is 0.105mg/ml, it is compatible with the commercial hepatitis B vaccine (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) with aluminum content of 0.35-0.62mg/ml). Compared with Kangtai), the produced antibody IgG and IgG2a subtype are better.
有益效果Beneficial effect
本发明的疫苗载体使用阴离子材料修饰氢氧化铝，稳定性更高，不易聚集，分散性好，更适合于临床使用。The vaccine carrier of the present invention uses anionic materials to modify aluminum hydroxide, has higher stability, is not easy to aggregate, has good dispersibility, and is more suitable for clinical use.
本发明的疫苗载体粒径为30-200nm，符合淋巴结递送的要求，能够有效地靶向淋巴结，实现疫苗的靶向递送。The vaccine carrier of the present invention has a particle size of 30-200 nm, which meets the requirements of lymph node delivery, can effectively target the lymph nodes, and realize the targeted delivery of the vaccine.
本发明的疫苗载体可被抗原提呈细胞高效摄取，有利于下一步免疫应答的发生。The vaccine carrier of the present invention can be efficiently taken up by the antigen-presenting cells, which is beneficial to the occurrence of the next immune response.
本发明的疫苗载体可以包载不同种类的抗原，且纳米粒本身有佐剂作用，可以帮助诱导抗原特异性的免疫反应，从而产生更强的免疫效果，二者具有协同作用。The vaccine carrier of the present invention can contain different kinds of antigens, and the nanoparticle itself has an adjuvant effect, which can help induce an antigen-specific immune response, thereby producing a stronger immune effect, and the two have a synergistic effect.
本发明的疫苗载体制备方法简单，无有机溶剂的加入，重复性好，稳定性高。The vaccine carrier of the invention has a simple preparation method, no organic solvent is added, good repeatability and high stability.
本发明的疫苗载体铝含量低，铝含量远远低于商品化铝凝胶(
Figure PCTCN2020136150-appb-000004
2％(美国InvivoGene公司)，可以减少金属离子蓄积的危险和局部的副作用，毒性低，提高了患者的适应性；同时在铝含量远远低于商品化铝凝胶吸附疫苗(重组乙型肝炎疫苗(酿酒酵母)深圳康泰)的情况下，诱导的免疫反应与商品化铝凝胶的免疫反应相当或更强，具有更好的免疫效果。 The vaccine carrier of the present invention has low aluminum content, which is far lower than commercial aluminum gel (
Figure PCTCN2020136150-appb-000004
2% (InvivoGene, USA), which can reduce the danger of metal ion accumulation and local side effects, has low toxicity, and improves the adaptability of patients; at the same time, the aluminum content is much lower than the commercial aluminum gel adsorption vaccine (recombinant hepatitis B In the case of the vaccine (Saccharomyces cerevisiae) Shenzhen Kangtai), the induced immune response is equivalent to or stronger than that of the commercial aluminum gel, which has a better immune effect.
相比现有技术，本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:
1，用阴离子聚合物材料和铝盐复合形成的氢氧化铝纳米粒作为疫苗载体，扩大了可用于包载铝盐的材料的范围，提高了普适性。1. Aluminum hydroxide nanoparticles formed by the composite of anionic polymer materials and aluminum salts are used as vaccine carriers, which expands the range of materials that can be used for encapsulating aluminum salts and improves the universality.
2，用阴离子聚合物材料和铝盐复合形成的氢氧化铝纳米粒作为疫苗载体，可以在使用无PEG修饰的材料时形成稳定的纳米粒，纳米粒分散性好， 稳定性高。2. Aluminum hydroxide nanoparticles formed by the composite of anionic polymer materials and aluminum salts are used as vaccine carriers, and stable nanoparticles can be formed when materials without PEG modification are used. The nanoparticles have good dispersibility and high stability.
3，本发明所用的阴离子聚合物材料具有良好的生物相容性，毒性小，安全性高。例如其中的硫酸软骨素，其是一种生物相容性很好的粘多糖，广泛存在于人和动物的软骨组织，作为一种食品用于治疗骨关节炎；同时FDA批准其可以作为的皮肤代替品，在我国也已有上市的硫酸软骨素片以及硫酸软骨素注射液，其安全性远远高于合成高分子材料，且价廉易得。3. The anionic polymer material used in the present invention has good biocompatibility, low toxicity and high safety. For example, chondroitin sulfate, which is a kind of mucopolysaccharide with good biocompatibility, is widely present in cartilage tissues of humans and animals. It is used as a food for the treatment of osteoarthritis; at the same time, the FDA approved it as a skin Alternatives, chondroitin sulfate tablets and chondroitin sulfate injections are also on the market in my country, which are much safer than synthetic polymer materials and are cheap and easy to obtain.
4，本发明方法简单，且超声和注射泵的方法可用于大规模生产，有利于工业转化，具有广阔的应用和市场前景。4. The method of the present invention is simple, and the method of ultrasound and syringe pump can be used for large-scale production, which is beneficial to industrial transformation and has broad application and market prospects.
附图说明Description of the drawings
图1为γ-聚谷氨酸-纳米粒粒径图。Figure 1 is a graph of the particle size of γ-polyglutamic acid-nanoparticles.
图2为聚谷氨酸接枝聚乙二醇-HBsAg纳米粒的粒径图。Figure 2 shows the particle size of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles.
图3为硫酸软骨素-纳米粒粒径图。Figure 3 is a diagram showing the particle size of chondroitin sulfate-nanoparticles.
图4为γ-聚谷氨酸-纳米粒电镜图。Figure 4 is an electron micrograph of γ-polyglutamic acid-nanoparticles.
图5为聚谷氨酸接枝聚乙二醇-HBsAg纳米粒的电镜图。Figure 5 is an electron micrograph of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles.
图6为硫酸软骨素-纳米粒电镜图。Figure 6 is an electron micrograph of chondroitin sulfate-nanoparticles.
图7为DC2.4和Raw264.7细胞对抗原和载抗原的γ-聚谷氨酸-纳米粒(PGA)的摄取情况。Figure 7 shows the uptake of antigen and antigen-carrying gamma-polyglutamic acid-nanoparticles (PGA) by DC2.4 and Raw264.7 cells.
图8为DC2.4和Raw264.7细胞对抗原和载抗原的硫酸软骨素-纳米粒(ASN)的摄取情况。Figure 8 shows the uptake of antigens and antigen-carrying chondroitin sulfate-nanoparticles (ASN) by DC2.4 and Raw264.7 cells.
图9为γ-聚谷氨酸-纳米粒(PGA)在淋巴结的滞留情况。Figure 9 shows the retention of γ-polyglutamic acid-nanoparticles (PGA) in lymph nodes.
图10为γ-聚谷氨酸-纳米粒(PGA)的CTL结果。Figure 10 shows the CTL results of γ-polyglutamic acid-nanoparticles (PGA).
图11为γ-聚谷氨酸-纳米粒(PGA)的免疫抗体。Figure 11 shows immune antibodies against γ-polyglutamic acid-nanoparticles (PGA).
图12为聚谷氨酸接枝聚乙二醇-HBsAg纳米粒(PGN)的免疫抗体。Figure 12 shows the immune antibody of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles (PGN).
图13为硫酸软骨素-纳米粒(ASN)的免疫抗体。Figure 13 shows immune antibodies against chondroitin sulfate-nanoparticles (ASN).
具体实施方式Detailed ways
以下实施例是对本发明的进一步说明，但绝不是对本发明范围的限制。下面参照实施例进一步详细阐述本发明，但是本领域技术人员应当理解，本发明并不限于这些实施例以及使用的制备方法。而且，本领域技术人员根据本发明的描述可以对本发明进行等同替换、组合、改良或修饰，但这些都将包括在本发明的范围内。The following examples are further descriptions of the present invention, but they are by no means limiting the scope of the present invention. The present invention will be further elaborated below with reference to the examples, but those skilled in the art should understand that the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art can make equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included in the scope of the present invention.
实施例1Example 1
基于γ-聚谷氨酸材料氢氧化铝纳米粒的制备：往345ul 80mmol/L PH为8的Hepes缓冲液中加入125ul 1mg/ml的γ-聚谷氨酸，混合均匀，吸取550ul 1.76mmol/L的硫酸铝溶液，加入上述溶液中，超声5min,功率为120w，即得。Preparation of aluminum hydroxide nanoparticles based on γ-polyglutamic acid material: add 125ul 1mg/ml γ-polyglutamic acid to 345ul 80mmol/L Hepes buffer with a pH of 8, mix well, and draw 550ul 1.76mmol/ The aluminum sulfate solution of L is added to the above-mentioned solution, sonicated for 5 minutes, and the power is 120w, and it is obtained.
实施例2Example 2
基于γ-聚谷氨酸材料氢氧化铝纳米粒的制备：往360ul 100mmol/L PH为8的Hepes缓冲液中加入120ul 8mg/ml的γ-聚谷氨酸，混合均匀，吸取555ul 17.56mmol/L的硫酸铝溶液，加入上述溶液中，超声10min,功率为150w，即得。Preparation of aluminum hydroxide nanoparticles based on γ-polyglutamic acid material: add 120ul 8mg/ml γ-polyglutamic acid to 360ul 100mmol/L Hepes buffer with a pH of 8, mix well, and draw 555ul 17.56mmol/ The aluminum sulfate solution of L is added to the above-mentioned solution, sonicated for 10 minutes, and the power is 150w, and it is obtained.
实施例3Example 3
基于γ-聚谷氨酸材料氢氧化铝纳米粒-OVA的制备：往370ul 100mmol/LPH为8的Hepes缓冲液中加入130ul 20mg/ml的γ-聚谷氨酸，混合均匀，450ul吸取2.16mmol/L的硫酸铝溶液和100ul 4mg/ml OVA溶液混合均匀，加入上述溶液中，超声8min,功率为100w，即得。Preparation of aluminum hydroxide nanoparticles-OVA based on γ-polyglutamic acid material: Add 130ul 20mg/ml γ-polyglutamic acid to 370ul 100mmol/LPH of 8 Hepes buffer, mix well, 450ul absorb 2.16mmol /L aluminum sulfate solution and 100ul 4mg/ml OVA solution are mixed uniformly, add to the above solution, ultrasonic 8min, power 100w, and it is obtained.
实施例4Example 4
基于γ-聚谷氨酸材料氢氧化铝纳米粒-OVA的制备：往400ul 70mmol/LPH为8的Hepes缓冲液中加入140ul 7.5mg/ml的γ-聚谷氨酸，混合均匀，吸取460ul 1.4mmol/L的硫酸铝溶液和100ul 1mg/ml OVA溶液混合均匀，加入上述溶液中，超声6min,功率为150w，即得。Preparation of aluminum hydroxide nanoparticles-OVA based on γ-polyglutamic acid material: add 140ul 7.5mg/ml γ-polyglutamic acid to 400ul 70mmol/LPH of 8 Hepes buffer, mix well, and draw 460ul 1.4 Mix the mmol/L aluminum sulfate solution and 100ul 1mg/ml OVA solution uniformly, add them to the above solution, sonicate for 6min, and the power is 150w, and it will be obtained.
实施例5Example 5
基于γ-聚谷氨酸材料氢氧化铝纳米粒-OVA-CpG的制备：往380ul 80mmol/L PH为8的Hepes缓冲液中加入110ul 10mg/ml的γ-聚谷氨酸，混合均匀，吸取500ul 2mmol/L的硫酸铝溶液和100ul 2mg/ml OVA溶液以及10ul 2mg/ml CpG溶液混合均匀，加入上述溶液中，超声5min,功率为120w，即得。Preparation of aluminum hydroxide nanoparticles-OVA-CpG based on the γ-polyglutamic acid material: Add 110ul 10mg/ml γ-polyglutamic acid to 380ul 80mmol/L Hepes buffer with a pH of 8, mix well, and absorb Mix 500ul 2mmol/L aluminum sulfate solution, 100ul 2mg/ml OVA solution and 10ul 2mg/ml CpG solution, add to the above solution, sonicate for 5min, power 120w, and get it.
实施例6Example 6
基于γ-聚谷氨酸材料氢氧化铝纳米粒-OVA-CpG的制备：往400ul 50mmol/L PH为8的Hepes缓冲液中加入100ul 5mg/ml的γ-聚谷氨酸，混合均匀，吸取560ul 1.5mmol/L的硫酸铝溶液和50ul 4mg/ml OVA溶液以及10ul 500ug/ml CpG溶液混合均匀，加入上述溶液中，超声15min,功率为100w，即得。Preparation of aluminum hydroxide nanoparticles-OVA-CpG based on γ-polyglutamic acid material: add 100ul 5mg/ml γ-polyglutamic acid to 400ul 50mmol/L Hepes buffer with a pH of 8, mix well, and absorb Mix 560ul 1.5mmol/L aluminum sulfate solution, 50ul 4mg/ml OVA solution and 10ul 500ug/ml CpG solution evenly, add to the above solution, sonicate for 15min, power 100w, and get it.
实施例7Example 7
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝纳米粒的制备：往340ul  100mmol/L PH为8的Hepes缓冲液中加入125ul 2.5mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取560ul 1.6mmol/L的硫酸铝溶液加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 125ul 2.5mg/ml polyglutamic acid grafted polyethylene glycol to 340ul 100mmol/L Hepes buffer with a pH of 8 Mix the materials evenly, add 560ul 1.6mmol/L aluminum sulfate solution to the above mixed solution, and vortex for 30s to get.
实施例8Example 8
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝纳米粒的制备：往360ul 80mmol/L PH为8的Hepes缓冲液中加入110ul 7.5mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取555ul 2mmol/L的硫酸铝溶液加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 110ul 7.5mg/ml polyglutamic acid grafted polyethylene glycol to 360ul 80mmol/L Hepes buffer with a pH of 8 Mix the materials evenly, add 555ul 2mmol/L aluminum sulfate solution to the above mixed solution, and vortex for 30s to get.
实施例9Example 9
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝纳米粒的制备：往350ul 100mmol/L PH为8的Hepes缓冲液中加入100ul 10mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取500ul 5.5mmol/L的硫酸铝溶液加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 100ul 10mg/ml polyglutamic acid grafted polyethylene glycol material to 350ul 100mmol/L Hepes buffer with a pH of 8 , Mix well, add 500ul 5.5mmol/L aluminum sulfate solution to the above mixed solution, vortex for 30s to get.
实施例10Example 10
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝-HBsAg纳米粒的制备：往345ul 100mmol/L PH为8的Hepes缓冲液中加入110ul 10mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取300ul 6.6mmol/L的硫酸铝溶液和220ul 50ug/ml HBsAg溶液混合均匀，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 110ul 10mg/ml of polyglutamic acid grafted polyethylene to 345ul 100mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 300ul 6.6mmol/L aluminum sulfate solution and 220ul 50ug/ml HBsAg solution and mix well, add to the above mixed solution, vortex for 30s to get.
实施例11Example 11
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝-HBsAg纳米粒的制备：往400ul 90mmol/L PH为8的Hepes缓冲液中加入125ul 10mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取290ul 9mmol/L的硫酸铝溶液和200ul 400ug/ml HBsAg溶液混合均匀，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 125ul 10mg/ml of polyglutamic acid grafted polyethylene to 400ul 90mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 290ul 9mmol/L aluminum sulfate solution and 200ul 400ug/ml HBsAg solution and mix evenly, add to the above mixed solution, vortex for 30s to get.
实施例12Example 12
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝-HBsAg纳米粒的制备：往400ul 100mmol/L PH为8的Hepes缓冲液中加入125ul 12mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取250ul 10mmol/L的硫酸铝溶液和250ul 20ug/ml HBsAg溶液混合均匀，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 125ul 12mg/ml of polyglutamic acid grafted polyethylene to 400ul 100mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 250ul 10mmol/L aluminum sulfate solution and 250ul 20ug/ml HBsAg solution and mix evenly, add to the above mixed solution, vortex for 30s to get.
实施例13Example 13
基于聚谷氨酸接枝聚乙二醇材料氢氧化铝-HBsAg纳米粒的制备：往380ul 90mmol/L PH为8的Hepes缓冲液中加入120ul 10mg/ml的聚谷氨酸接枝聚乙二醇材料，混合均匀，吸取280ul 8mmol/L的硫酸铝溶液和240ul 10ug/ml HBsAg溶液混合均匀，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide-HBsAg nanoparticles based on polyglutamic acid grafted polyethylene glycol material: add 120ul 10mg/ml of polyglutamic acid grafted polyethylene to 380ul 90mmol/L Hepes buffer with a pH of 8 Alcohol material, mix well, suck 280ul 8mmol/L aluminum sulfate solution and 240ul 10ug/ml HBsAg solution and mix evenly, add to the above mixed solution, vortex for 30s to get.
实施例14Example 14
基于硫酸软骨素氢氧化铝纳米粒的制备：往340ul 100mmol/L PH为7.6的Hepes缓冲液中加入220ul 10mg/ml的硫酸软骨素材料，混合均匀，吸取400ul 10mmol/L的硫酸铝溶液，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide nanoparticles based on chondroitin sulfate: add 220ul 10mg/ml chondroitin sulfate material to 340ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, suck 400ul 10mmol/L aluminum sulfate solution, add In the above mixed solution, vortex for 30 seconds.
实施例15Example 15
基于硫酸软骨素氢氧化铝纳米粒的制备：往400ul 100mmol/L PH为7.6的Hepes缓冲液中加入200ul 10mg/ml的硫酸软骨素材料，混合均匀，吸取380ul 10mmol/L的硫酸铝溶液，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide nanoparticles based on chondroitin sulfate: add 200ul 10mg/ml chondroitin sulfate material to 400ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, suck 380ul 10mmol/L aluminum sulfate solution, add In the above mixed solution, vortex for 30 seconds.
实施例16Example 16
基于硫酸软骨素氢氧化铝-OVA纳米粒的制备：往400ul 100mmol/L PH为7.6的Hepes缓冲液中加入300ul 10mg/ml的硫酸软骨素材料，混合均匀，吸取340ul 10mmol/L的硫酸铝溶液和60ul 1mg/ml的OVA溶液混合均匀，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide-OVA nanoparticles based on chondroitin sulfate: Add 300ul 10mg/ml chondroitin sulfate material to 400ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, and draw 340ul 10mmol/L aluminum sulfate solution Mix well with 60ul 1mg/ml OVA solution, add to the above mixture, vortex for 30s to get.
实施例17Example 17
基于硫酸软骨素氢氧化铝-OVA-CpG纳米粒的制备：往360ul 100mmol/L PH为7.6的Hepes缓冲液中加入280ul 10mg/ml的硫酸软骨素材料，混合均匀，吸取350ul 10mmol/L的硫酸铝溶液和70ul 0.85mg/ml的OVA溶液以及20ul 2mg/ml CpG溶液混合均匀，加入上述混合液中，涡旋30s即得。Preparation of aluminum hydroxide-OVA-CpG nanoparticles based on chondroitin sulfate: add 280ul 10mg/ml chondroitin sulfate material to 360ul 100mmol/L Hepes buffer with a pH of 7.6, mix well, and absorb 350ul 10mmol/L sulfuric acid Mix the aluminum solution with 70ul 0.85mg/ml OVA solution and 20ul 2mg/ml CpG solution evenly, add to the above mixture, and vortex for 30s to get.
实施例18Example 18
基于硫酸软骨素氢氧化铝-OVA纳米粒的制备：往20ml 100mmol/L PH为7.8的Hepes缓冲液中加入10ml 10mg/ml的硫酸软骨素材料以及3ml的1mg/ml OVA溶液，混合均匀加入1号注射器，33ml 6.06mmol/L硫酸铝溶液加入2号注射器，两个注射器同时通过微量注射泵以20ml/min的速度通过异型三通道微流体装置，收集混合后的液体，即为纳米粒。Preparation of aluminum hydroxide-OVA nanoparticles based on chondroitin sulfate: add 10ml 10mg/ml chondroitin sulfate material and 3ml 1mg/ml OVA solution to 20ml 100mmol/L Hepes buffer with a pH of 7.8, mix well and add 1 No. 2 syringe, 33ml 6.06mmol/L aluminum sulfate solution is added to No. 2 syringe, and the two syringes are simultaneously passed through the micro-syringe pump at a speed of 20ml/min through the special-shaped three-channel microfluidic device, and the mixed liquid is collected, which is the nanoparticle.
实施例19Example 19
基于硫酸软骨素氢氧化铝-OVA纳米粒的制备：往20ml 100mmol/L PH为7.8的Hepes缓冲液中加入10ml 10mg/ml的硫酸软骨素材料以及3ml的1mg/ml OVA溶液，混合均匀加入1号注射器，33ml 4.04mmol/L硫酸铝溶液加入2号注射器，两个注射器同时通过微量注射泵以50ml/min的速度通过异型三通道微流体装置，收集混合后的液体，即为纳米粒。Preparation of Chondroitin Sulfate Aluminum Hydroxide-OVA Nanoparticles: Add 10ml 10mg/ml chondroitin sulfate material and 3ml 1mg/ml OVA solution to 20ml 100mmol/L Hepes buffer with a pH of 7.8, mix evenly and add 1 No. 2 syringe, 33ml 4.04mmol/L aluminum sulfate solution is added to No. 2 syringe. Both syringes pass through a micro-syringe pump at a speed of 50ml/min through a special-shaped three-channel microfluidic device to collect the mixed liquid, which is the nanoparticle.
实施例20Example 20
纳米粒粒径测定：使用Zetasizer Nano ZS90激光粒度分析仪测定实施 例1-19的γ-聚谷氨酸-纳米粒、聚谷氨酸接枝聚乙二醇-HBsAg、硫酸软骨素-纳米粒的粒径分布，分别取1ml的实施例1-19的纳米粒溶液，将样品放入样品池内，测定温度设为25℃，其结果如表一所示，结果显示纳米粒粒径在100nm左右，PDI符合要求，分布均一。Nanoparticle size measurement: use Zetasizer Nano ZS90 laser particle size analyzer to measure the γ-polyglutamic acid-nanoparticles, polyglutamic acid grafted polyethylene glycol-HBsAg, and chondroitin sulfate-nanoparticles of Examples 1-19 Take 1ml of the nanoparticle solution of Example 1-19, put the sample into the sample cell, and set the measurement temperature to 25℃. The results are shown in Table 1. The results show that the nanoparticle size is about 100nm. , PDI meets the requirements and the distribution is uniform.
表1 1-19实施例的粒径测定结果Table 1 Particle size measurement results of Examples 1-19
实施例编号Example number Size(nm)Size(nm) PDI PDI
11 104.7104.7 0.1300.130
22 174.3174.3 0.2300.230
33 154.2154.2 0.2320.232
44 123.4123.4 0.1680.168
55 129.9129.9 0.1800.180
66 145.4145.4 0.2510.251
77 107.8107.8 0.1820.182
88 115.5115.5 0.2020.202
99 79.379.3 0.2070.207
1010 173.7173.7 0.2410.241
1111 113.4113.4 0.1900.190
1212 164.0164.0 0.2120.212
1313 117.3117.3 0.1950.195
1414 109.0109.0 0.1960.196
1515 118.5118.5 0.2110.211
1616 106.2106.2 0.2260.226
1717 181.0181.0 0.2350.235
1818 146.0146.0 0.2300.230
1919 131.2131.2 0.2370.237
实施例21Example 21
γ-聚谷氨酸-纳米粒的粒径测定：使用Zetasizer Nano ZS90激光粒度分析仪测定实施例1的γ-聚谷氨酸-纳米粒的粒径分布。取1ml的纳米粒溶液，将样品放入样品池内，测定温度设为25℃，结果如图1所示。纳米粒粒径在100nm左右，分布均一。Measurement of the particle size of γ-polyglutamic acid-nanoparticles: The particle size distribution of the γ-polyglutamic acid-nanoparticles of Example 1 was measured using a Zetasizer Nano ZS90 laser particle size analyzer. Take 1ml of the nanoparticle solution, put the sample into the sample cell, and set the measurement temperature to 25°C. The result is shown in Figure 1. The size of the nanoparticles is about 100nm, and the distribution is uniform.
实施例22Example 22
聚谷氨酸接枝聚乙二醇-HBsAg纳米粒的粒径测定：使用Zetasizer Nano ZS90激光粒度分析仪测定实施例11的聚谷氨酸接枝聚乙二醇-HBsAg纳米粒的粒径分布。取1ml的纳米粒溶液，将样品放入样品池内，测定温度设为25℃，结果如图2所示。纳米粒粒径在100nm左右，分布均一。Determination of the particle size of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles: The particle size distribution of the polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles of Example 11 was measured using a Zetasizer Nano ZS90 laser particle size analyzer . Take 1ml of the nanoparticle solution, put the sample into the sample cell, and set the measurement temperature to 25°C. The result is shown in Figure 2. The size of the nanoparticles is about 100nm, and the distribution is uniform.
实施例23Example 23
硫酸软骨素-纳米粒的粒径测定：使用Zetasizer Nano ZS90激光粒度分析仪测定实施例16的硫酸软骨素-纳米粒的粒径分布。取1ml的纳米粒溶液，将样品放入样品池内，测定温度设为25℃，结果如图3所示。纳米粒粒径在100nm左右，分布均一。Determination of the particle size of chondroitin sulfate-nanoparticles: The particle size distribution of the chondroitin sulfate-nanoparticles of Example 16 was measured using a Zetasizer Nano ZS90 laser particle size analyzer. Take 1ml of the nanoparticle solution, put the sample into the sample cell, and set the measurement temperature to 25°C. The result is shown in Figure 3. The size of the nanoparticles is about 100nm, and the distribution is uniform.
实施例24Example 24
γ-聚谷氨酸-纳米粒照透射电镜：将实施例1的γ-聚谷氨酸-纳米粒样品置于铜网上，静置5min，然后用磷钨酸染色1min，之后用滤纸吸走铜网上多余的染液，室温晾干样品，在200kv条件下，透射电镜观察样品。结果如图4所示，由实验结果可知纳米粒均为圆整的颗粒，粒径在100nm以下。γ-Polyglutamic Acid-Nanoparticles Photo Transmission Electron Microscope: Place the γ-Polyglutamic Acid-Nanoparticles sample of Example 1 on a copper mesh, let stand for 5 minutes, then stain with phosphotungstic acid for 1 minute, and then suck it away with filter paper Excess dye solution on the copper mesh, dry the sample at room temperature, and observe the sample under a transmission electron microscope at 200kv. The results are shown in Fig. 4, and it can be seen from the experimental results that the nanoparticles are all round particles with a particle size below 100 nm.
实施例25Example 25
聚谷氨酸接枝聚乙二醇-HBsAg纳米粒照透射电镜：将实施例11的聚谷氨酸接枝聚乙二醇-HBsAg纳米粒样品置于铜网上，静置5min，然后用磷钨酸染色1min，之后用滤纸吸走铜网上多余的染液，室温晾干样品，在200kv条件下，透射电镜观察样品。结果如图5所示，由实验结果可知纳米粒均为圆整的颗粒，粒径在100nm以下。Transmission electron microscopy of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles: the sample of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles of Example 11 was placed on a copper mesh, and then allowed to stand for 5 min. Tungstic acid is stained for 1 min, and then the excess dye solution on the copper net is sucked away with filter paper, and the sample is dried at room temperature. Under the condition of 200kv, the sample is observed by transmission electron microscope. The results are shown in Fig. 5, and it can be seen from the experimental results that the nanoparticles are all round particles with a particle size below 100 nm.
实施例26Example 26
硫酸软骨素-纳米粒照透射电镜：将实施例16的硫酸软骨素-纳米粒样品置于铜网上，静置5min，然后用磷钨酸染色1min，之后用滤纸吸走铜网上多余的染液，室温晾干样品，在200kv条件下，透射电镜观察样品。结果如图6所示，由实验结果可知纳米粒均为圆整的颗粒，粒径在100nm以下。Chondroitin sulfate-nanoparticles according to transmission electron microscopy: the chondroitin sulfate-nanoparticles sample of Example 16 was placed on a copper mesh, allowed to stand for 5 min, and then stained with phosphotungstic acid for 1 min, and then filter paper was used to absorb the excess dye solution on the copper mesh , Dry the sample at room temperature, and observe the sample under a transmission electron microscope under 200kv. The results are shown in Fig. 6, and it can be seen from the experimental results that the nanoparticles are all round particles with a particle size below 100 nm.
实施例27Example 27
γ-聚谷氨酸-纳米粒在DC2.4和Raw264.7细胞的摄取：在十二孔板中，每孔种入1×10 6个DC2.4或Raw264.7两种细胞，放入孵箱4～6小时，细胞贴壁后，向每孔中加入50μl FITC标记的OVA，或用FITC标记的OVA制备的实施例1的γ-聚谷氨酸-纳米粒。37度条件下摄取1小时后，弃去上清液，用PBS轻轻冲洗细胞表面2次，再用胰酶消化细胞(DC2.4直接吹打)，2000rpm 3min离心洗涤细胞2次，最后用400μl PBS重悬细胞，用流式细胞仪检测。结果如图7a和7b所示，γ-聚谷氨酸-纳米粒可被抗原呈递细胞高效地摄取，在DC2.4细胞上的摄取率远远高于游离的OVA，具有显著性差异(P<0.0001)；在Raw264.7细胞上的摄取率远远高于游离的OVA，具有显著性差异(P<0.001)，可见通过纳米粒包裹抗原显著提高了抗原的摄取效率。 Uptake of γ-polyglutamic acid-nanoparticles in DC2.4 and Raw264.7 cells: In a twelve-well plate, plant 1×10 6 DC2.4 or Raw264.7 cells in each well, and put them in Incubate for 4-6 hours, after the cells adhere to the wall, add 50 μl of FITC-labeled OVA or the γ-polyglutamic acid-nanoparticle of Example 1 prepared with FITC-labeled OVA to each well. After ingesting at 37°C for 1 hour, discard the supernatant, gently wash the cell surface twice with PBS, then trypsinize the cells (DC2.4 direct pipetting), wash the cells twice by centrifugation at 2000rpm for 3min, and finally use 400μl The cells were resuspended in PBS and tested by flow cytometry. The results are shown in Figures 7a and 7b. γ-polyglutamic acid-nanoparticles can be efficiently taken up by antigen-presenting cells, and the uptake rate on DC2.4 cells is much higher than that of free OVA, which has a significant difference (P <0.0001); The uptake rate on Raw264.7 cells is much higher than that of free OVA, with a significant difference (P<0.001). It can be seen that the antigen uptake efficiency is significantly improved by nanoparticle-encapsulated antigen.
实施例28Example 28
硫酸软骨素-纳米粒在DC2.4和Raw264.7细胞的摄取：在十二孔板中，每孔种入1×10 6个DC2.4或Raw264.7两种细胞，放入孵箱4～6小时，细胞贴壁后，向每孔中加入50μl FITC-OVA，或用FITC标记的OVA制备的实施例16的硫酸软骨素-纳米粒，37度条件下摄取1小时后，弃去上清液，用PBS轻轻冲洗细胞表面2次，再用胰酶消化细胞(DC2.4直接吹打)，2000rpm 3min离心洗涤细胞2次，最后用400μl PBS重悬细胞，用流式细胞仪检测。结果如图8a和8b所示，硫酸软骨素-纳米粒可被抗原呈递细胞高效摄取，在DC2.4细胞上的摄取率远远高于游离的OVA，具有显著性差异(P<0.0001)；在Raw264.7细胞上的摄取率远远高于游离的OVA，具有显著性差异(P<0.0001)，可见通过纳米粒包裹抗原显著提高了免疫细胞对抗原的摄取效率。 Uptake of chondroitin sulfate-nanoparticles in DC2.4 and Raw264.7 cells: In a twelve-well plate, plant 1×10 6 cells of DC2.4 or Raw264.7 in each well, and put them in the incubator 4 ~6 hours, after the cells adhere to the wall, add 50μl FITC-OVA or the chondroitin sulfate-nanoparticle of Example 16 prepared with FITC-labeled OVA to each well. After ingesting at 37°C for 1 hour, discard it. The clear solution was washed with PBS gently on the cell surface twice, then the cells were trypsinized (DC2.4 direct pipetting), and the cells were washed twice by centrifugation at 2000 rpm for 3 min. Finally, the cells were resuspended in 400 μl PBS and detected by flow cytometry. The results are shown in Figures 8a and 8b. Chondroitin sulfate-nanoparticles can be efficiently taken up by antigen-presenting cells, and the uptake rate on DC2.4 cells is much higher than that of free OVA, with a significant difference (P<0.0001); The uptake rate on Raw264.7 cells is much higher than that on free OVA, with a significant difference (P<0.0001). It can be seen that the antigen uptake efficiency of immune cells is significantly improved by nanoparticle-encapsulated antigen.
实施例29Example 29
γ-聚谷氨酸-纳米粒在淋巴结的滞留研究：对C57BL/6小鼠脚掌注射25μl FITC标记的OVA制备的实施例1的γ-聚谷氨酸-纳米粒溶液，于给药4小时和20小时后,断颈处死小鼠，分离腘弯处淋巴结。用1ml注射器针头适当刺破淋巴结，置于1mg/ml D型胶原酶溶液中37℃消化30分钟，于细胞筛网上研磨淋巴结，得到的细胞离心洗涤2遍，50μl流式染色缓冲液重悬细胞，加入50μl含1μg抗小鼠CD11c PE抗体的流式染色缓冲液，吹打均匀，4℃染色40分钟，离心洗涤细胞2遍，400μlPBS重悬，流式细胞仪检测。结果如图9a所示，纳米粒在4小时就已到达淋巴结，约5％FITC阳性细胞被检测到，说明γ-聚谷氨酸-纳米粒具有淋巴结靶向能力。20小时时，淋巴结内仍能检测到约1％FITC阳性细胞，说明γ-聚谷氨酸-纳米粒在淋巴结中体现了一定的滞留能力。流式抗体对DC细胞特征表面分子CD11c的染色，从淋巴结细胞群中区分出了DC细胞，其中FITC+CD11c+双阳性信号即为摄取了γ-聚谷氨酸纳米粒的DC细胞，如图9b所示，在淋巴结内大部分纳米粒是被DC细胞摄取的，这也为下一步的免疫应答的发生提供了有利条件。Study on the retention of γ-polyglutamic acid-nanoparticles in lymph nodes: C57BL/6 mice were injected with 25μl FITC-labeled OVA into the γ-polyglutamic acid-nanoparticle solution of Example 1 and administered for 4 hours Twenty hours later, the mice were sacrificed by cervical dislocation, and the lymph nodes at the popliteal curve were separated. Use a 1ml syringe needle to properly puncture the lymph nodes, place them in 1mg/ml D collagenase solution at 37°C for 30 minutes, grind the lymph nodes on a cell sieve, and wash the cells twice by centrifugation, and resuspend the cells in 50μl flow staining buffer , Add 50μl of flow cytometry staining buffer containing 1μg of anti-mouse CD11c PE antibody, pipette evenly, stain for 40 minutes at 4°C, centrifuge to wash the cells twice, resuspend in 400μl PBS, and detect by flow cytometry. The results are shown in Figure 9a. The nanoparticles reached the lymph nodes within 4 hours, and about 5% of FITC-positive cells were detected, indicating that the γ-polyglutamic acid-nanoparticles have lymph node targeting ability. At 20 hours, about 1% of FITC-positive cells could still be detected in the lymph nodes, indicating that the γ-polyglutamic acid-nanoparticles exhibited a certain retention ability in the lymph nodes. The flow cytometry antibody stained the characteristic surface molecule CD11c of DC cells, distinguishing DC cells from the lymph node cell population. Among them, the FITC+CD11c+ double positive signal is the DC cells that have taken up γ-polyglutamic acid nanoparticles, as shown in Figure 9b As shown, most of the nanoparticles in the lymph nodes are taken up by DC cells, which also provides favorable conditions for the occurrence of the next immune response.
实施例30Example 30
γ-聚谷氨酸-纳米粒的细胞毒性T淋巴细胞(CTL)实验和免疫抗体检测：第0天和第7天免疫，小鼠脚掌注射25ul的实施例5的γ-聚谷氨酸-纳米粒(每只5ugOVA,0.5ug CpG)，第14天通过CFSE染色的方法检测体内CTL反应。结果如图10所示，γ-聚谷氨酸-纳米粒可以产生较强的抗原特异性细 胞免疫反应，其CTL显著高于游离OVA+CpG组,具有显著性差异(P<0.001)。Cytotoxic T lymphocyte (CTL) test and immune antibody detection of γ-polyglutamic acid-nanoparticles: On day 0 and day 7, mice were injected with 25ul of γ-polyglutamic acid from Example 5 on the soles of their feet. Nanoparticles (each 5ugOVA, 0.5ug CpG), on the 14th day by CFSE staining method to detect in vivo CTL response. The results are shown in Figure 10, γ-polyglutamic acid-nanoparticles can produce a strong antigen-specific cellular immune response, and its CTL is significantly higher than that of the free OVA+CpG group, with a significant difference (P<0.001).
实施例31Example 31
γ-聚谷氨酸-纳米粒的免疫抗体检测：0，7，14天免疫，小鼠脚掌注射25ul的实施例5的γ-聚谷氨酸-纳米粒，(每只5ugOVA,0.5ug CpG)，第21天眼眶取血，检测血清中OVA特异性抗体。结果如图11所示，其中图11a、图11b和图11c分别是IgG、IgG1和IgG2a的抗体检测结果。由实验结果可知，γ-聚谷氨酸-纳米粒可以产生较强的抗原特异性免疫反应，IgG水平显著高于游离的OVA+CpG组，具有显著性差异(P<0.001)，表征Th1型免疫应答的IgG2a水平也显著高于游离的OVA+CpG组，具有显著性差异(P<0.01)。Immune antibody detection of γ-polyglutamic acid-nanoparticles: 0, 7, and 14 days of immunization, mice were injected with 25ul of the γ-polyglutamic acid-nanoparticles of Example 5 in the sole of the foot, (each 5ugOVA, 0.5ug CpG ), blood was taken from the orbit on the 21st day, and OVA-specific antibodies in the serum were detected. The results are shown in Figure 11, where Figure 11a, Figure 11b and Figure 11c are the antibody detection results of IgG, IgG1 and IgG2a, respectively. It can be seen from the experimental results that γ-polyglutamic acid-nanoparticles can produce a strong antigen-specific immune response, and the IgG level is significantly higher than that of the free OVA+CpG group, with a significant difference (P<0.001), which characterizes the Th1 type The IgG2a level of the immune response was also significantly higher than that of the free OVA+CpG group, with a significant difference (P<0.01).
实施例32Example 32
聚谷氨酸接枝聚乙二醇-HBsAg纳米粒的免疫抗体检测：第0，14，28天免疫，小鼠脚掌分别注射25ul的实施例11的聚谷氨酸接枝聚乙二醇-HBsAg，其剂量按每只小鼠2ugHBsAg，35天眼眶取血，检测血清中HBsAg特异性抗体。结果如图12，其中Free代表游离抗原组，Vaccine代表市售疫苗组(重组乙型肝炎疫苗(酿酒酵母)深圳康泰,生产批号：B201701001)，PGN代表聚谷氨酸接枝聚乙二醇-HBsAg纳米粒组，图12a、图12b和图12c三个图分别是IgG、IgG1和IgG2a两种亚型的抗体检测结果。由实验结果可知，聚谷氨酸接枝聚乙二醇-HBsAg纳米粒可以产生较强的抗原特异性免疫反应，IgG水平显著高于游离的抗原组(P<0.0001)和市售疫苗组(P<0.001),IgG2a水平也显著高于游离抗原组(P<0.0001)和市售疫苗组(P<0.01)；IgG1水平显著高于游离抗原组(P<0.01)，和市售疫苗组相当。聚谷氨酸接枝聚乙二醇-HBsAg纳米粒能够诱导比游离抗原和市售商品化铝凝胶吸附疫苗更强的抗体水平，说明其本身具有一定的佐剂作用。Immune antibody detection of polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles: on the 0th, 14th, and 28th day, mice were injected with 25ul of the polyglutamic acid grafted polyethylene glycol of Example 11 in the soles of the mice. The dose of HBsAg was 2ugHBsAg per mouse. Blood was taken from the orbit at 35 days to detect HBsAg-specific antibodies in the serum. The results are shown in Figure 12, where Free represents the free antigen group, Vaccine represents the commercially available vaccine group (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen Kangtai, production batch number: B201701001), and PGN represents polyglutamic acid grafted polyethylene glycol- HBsAg nanoparticle group, Figure 12a, Figure 12b and Figure 12c are the antibody detection results of the two subtypes of IgG, IgG1 and IgG2a, respectively. It can be seen from the experimental results that polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles can produce a strong antigen-specific immune response, and the IgG level is significantly higher than that of the free antigen group (P<0.0001) and the commercially available vaccine group ( P<0.001), the IgG2a level was also significantly higher than the free antigen group (P<0.0001) and the commercial vaccine group (P<0.01); the IgG1 level was significantly higher than the free antigen group (P<0.01), which was comparable to the commercial vaccine group . Polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles can induce stronger antibody levels than free antigen and commercial aluminum gel adsorption vaccines, indicating that they have a certain adjuvant effect.
实施例33Example 33
硫酸软骨素-纳米粒的免疫抗体检测：第0、7、14天免疫，小鼠脚掌注射25ul的实施例17的硫酸软骨素-纳米粒，(每只1.5ugOVA,1ug CpG)，第21天眼眶取血，检测血清中OVA特异性抗体。结果如图13所示，其中图13a、图13b和图13c三个图分别是IgG、IgG1和IgG2a两种亚型的抗体检测结果。由实验结果可知，硫酸软骨素-纳米粒可以产生较强的抗原特异性免疫反应，IgG水平显著高于游离的OVA+CpG组，具有显著性差异(P<0.01)，IgG1和IgG2a水平也显著高于游离的OVA+CpG组(P<0.05)。Immune antibody detection of chondroitin sulfate-nanoparticles: immunization on days 0, 7, and 14, mice were injected with 25ul of chondroitin sulfate-nanoparticles of Example 17 on the soles of the feet (1.5ugOVA, 1ug CpG), day 21 Blood was taken from the orbit to detect OVA-specific antibodies in the serum. The results are shown in Figure 13, where Figure 13a, Figure 13b, and Figure 13c are the results of the antibody detection of the two subtypes of IgG, IgG1 and IgG2a, respectively. It can be seen from the experimental results that chondroitin sulfate-nanoparticles can produce a strong antigen-specific immune response, and the IgG level is significantly higher than that of the free OVA+CpG group, with a significant difference (P<0.01), and the IgG1 and IgG2a levels are also significant Higher than the free OVA+CpG group (P<0.05).
以上所述仅是本发明的优选实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以优选实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容做出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed in the preferred embodiments as above, it is not intended to limit the present invention. Anyone skilled in the art, Without departing from the scope of the technical solution of the present invention, when the technical content disclosed above can be used to make some changes or modification into equivalent embodiments with equivalent changes, but any content that does not deviate from the technical solution of the present invention, according to the present invention Technical essence Any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solutions of the present invention.

Claims (10)

  1. 一种基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，使用阴离子聚合物或其衍生物材料和铝盐复合形成疫苗载体，同时包载抗原。An aluminum salt nanoparticle vaccine carrier based on an anionic polymer and its derivatives is characterized in that an anionic polymer or its derivative material and an aluminum salt are used to form a vaccine carrier while simultaneously encapsulating an antigen.
  2. 如权利要求1所述的阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其中，所述铝盐为硫酸铝，基于重量份计，阴离子聚合物材料：硫酸铝为0.06～4.8:0.16-6.6。The aluminum salt nanoparticle vaccine carrier of an anionic polymer and its derivatives according to claim 1, wherein the aluminum salt is aluminum sulfate, and based on parts by weight, the anionic polymer material: aluminum sulfate is 0.06 to 4.8:0.16 -6.6.
  3. 如权利要求1-2任一所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，所述的阴离子聚合物材料包括天然或者内源性，半合成衍生，或者全合成的阴离子聚合物材料。The aluminum salt nanoparticle vaccine carrier based on anionic polymers and derivatives thereof according to any one of claims 1-2, wherein the anionic polymer material includes natural or endogenous, semi-synthetic derived, or Fully synthetic anionic polymer material.
  4. 如权利要求3所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，所述天然或者内源性阴离子聚合物材料包括：γ-聚谷氨酸、粘多糖，聚甘露糖醛酸，聚古罗糖醛酸，透明质酸，软骨素，肝素、角质素、海藻酸、葡聚糖、硫酸木甘露聚糖、岩藻多糖、岩藻半乳聚糖、海藻酸盐、琼脂、吉兰糖胶、印度树胶、卡拉亚胶、黄耆胶、兰胶、黄原胶、角叉菜胶的一种或多种；所述半合成衍生阴离子聚合物材料包括：硫酸肝素、硫酸软骨素、硫酸角质素、硫酸葡聚糖、羧甲基纤维素、交联焦糖、羧甲基淀粉、羧甲基葡聚糖、羧甲基壳聚糖、透明质酸衍生物、硫酸鼠李多糖、硫酸纤维素、硫酸凝胶多糖和磷酸壳聚糖的一种或多种；所述全合成阴离子聚合物材料包括：聚阴离子多肽、聚丙烯酸、聚甲基丙烯酸、聚谷氨酸、聚天冬氨酸、聚天冬氨酸接枝聚乙二醇、聚谷氨酸接枝聚乙二醇、聚卡波菲尔、羧乙烯聚合物、马来酸酐共聚物、硫醇化聚丙烯酸酯的一种或多种。The aluminum salt nanoparticle vaccine carrier based on anionic polymer and its derivatives according to claim 3, wherein the natural or endogenous anionic polymer material comprises: γ-polyglutamic acid, mucopolysaccharide, Polymannuronic acid, polyguluronic acid, hyaluronic acid, chondroitin, heparin, keratan, alginic acid, dextran, xylomannan sulfate, fucoidan, fucogalactan, seaweed One or more of acid salt, agar, gellan gum, Indian gum, carrageenan, tragacanth, blue gum, xanthan gum, and carrageenan; the semi-synthetic derivative anionic polymer material includes: Heparin sulfate, chondroitin sulfate, keratan sulfate, dextran sulfate, carboxymethyl cellulose, cross-linked caramel, carboxymethyl starch, carboxymethyl dextran, carboxymethyl chitosan, hyaluronic acid derivative One or more of sulphate, rhamnose sulfate, cellulose sulphate, curdlan sulphate and chitosan phosphate; the fully synthetic anionic polymer material includes: polyanionic polypeptide, polyacrylic acid, polymethacrylic acid, poly Glutamic acid, polyaspartic acid, polyaspartic acid grafted polyethylene glycol, polyglutamic acid grafted polyethylene glycol, polycarbophil, carboxyvinyl polymer, maleic anhydride copolymer, One or more thiolated polyacrylates.
  5. 如权利要求1-2任一所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，包括对阴离子聚合物材料进行衍生化修饰，包括PEG，羧基，羧甲基，硫酸根、亚硫酸根、磷酸根或亚磷酸根的修饰。The aluminum salt nanoparticle vaccine carrier based on anionic polymers and derivatives thereof according to any one of claims 1-2, which is characterized in that it comprises derivatizing and modifying anionic polymer materials, including PEG, carboxyl, carboxymethyl , Modification of sulfate, sulfite, phosphate or phosphite.
  6. 如权利要求1-2任一所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，所述的阴离子聚合物是线性聚合物，交联聚合物，或支化共聚物。The aluminum salt nanoparticle vaccine carrier based on anionic polymers and derivatives thereof according to any one of claims 1-2, wherein the anionic polymer is a linear polymer, a cross-linked polymer, or a branched polymer. Copolymer.
  7. 如权利要求1-2任一所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，阴离子聚合物及其衍生物的铝盐纳米粒通过直接吸附作用与抗原形成疫苗载体。The aluminum salt nanoparticle vaccine carrier based on the anionic polymer and its derivatives according to any one of claims 1-2, wherein the aluminum salt nanoparticles of the anionic polymer and its derivatives are formed by direct adsorption with the antigen. Vaccine carrier.
  8. 如权利要求1-2任一所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体，其特征在于，所述抗原选自：蛋白质抗原：甲型肝炎，乙型 肝炎或丙型肝炎抗原、破伤风类毒素、人乳头瘤病毒、白喉毒素、霍乱毒素、百日咳毒素、乙型脑炎病毒、流感病毒、结核、单纯疱疹病毒、麻疹病毒、风疹病毒、腮腺炎病毒、埃博拉病毒、狂犬病毒、呼吸道合胞病毒、西尼罗病毒、巨细胞病毒、疟疾抗原、肺炎链球菌、侵肺军团菌、脑膜炎奈瑟菌、铜绿假单胞菌、霍乱弧菌、A组链球菌抗原、或者其他重组蛋白抗原；免疫原性较弱的蛋白抗原，包括：牛血清白蛋白、溶菌酶、转铁蛋白、胰岛素、乳白蛋白、肌白蛋白、豆白蛋白、麦白蛋白、肌红蛋白、胶原蛋白、纤层蛋白；多肽抗原，包括：TRP2、HGP100、p15E、E6、E7、SIINFEKL、乙肝抗原表位肽S28-39、或者其他合成的多肽抗原及含有几种多肽序列的长多肽抗原；病毒或细菌裂解液抗原、病毒或细菌外膜囊泡抗原、肿瘤细胞裂解液抗原，肿瘤细胞膜囊泡抗原、肿瘤细胞外泌体抗原或者肿瘤模式抗原鸡卵清白蛋白(OVA)的一种或多种。The aluminum salt nanoparticle vaccine carrier based on anionic polymers and derivatives thereof according to any one of claims 1-2, wherein the antigen is selected from: protein antigens: hepatitis A, hepatitis B or C Hepatitis antigen, tetanus toxoid, human papilloma virus, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, herpes simplex virus, measles virus, rubella virus, mumps virus, Ebola Virus, rabies virus, respiratory syncytial virus, West Nile virus, cytomegalovirus, malaria antigen, Streptococcus pneumoniae, Legionella pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, group A chain Coccus antigens, or other recombinant protein antigens; protein antigens with weak immunogenicity, including: bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myalbumin, soy albumin, wheat albumin, muscle Red protein, collagen, laminin; peptide antigens, including: TRP2, HGP100, p15E, E6, E7, SIINFEKL, hepatitis B epitope peptide S28-39, or other synthetic peptide antigens and long peptides containing several peptide sequences Polypeptide antigen; virus or bacterial lysate antigen, virus or bacterial outer membrane vesicle antigen, tumor cell lysate antigen, tumor cell membrane vesicle antigen, tumor cell exosomal antigen or tumor model antigen chicken ovalbumin (OVA) one Kind or more.
  9. 如权利要求1-8任一所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体的制备方法，其特征在于，包括以下步骤：The method for preparing aluminum salt nanoparticle vaccine carrier based on anionic polymer and its derivatives according to any one of claims 1-8, characterized in that it comprises the following steps:
    (1)向Hepes缓冲液中加入阴离子聚合物或其衍生物材料，混合均匀，(1) Add the anionic polymer or its derivative materials to the Hepes buffer, mix well,
    (2)将铝盐硫酸铝和抗原溶液混合均匀，加入上述溶液中，(2) Mix the aluminum salt aluminum sulfate and the antigen solution uniformly, add to the above solution,
    (3)涡旋、超声或者通过微量注射泵进行混合。(3) Mix by vortexing, ultrasonic or micro-syringe pump.
  10. 如权利要求9所述的基于阴离子聚合物及其衍生物的铝盐纳米粒疫苗载体的制备方法，其特征在于，超声的功率为50-300W,时间为1-30min。The preparation method of aluminum salt nanoparticle vaccine carrier based on anionic polymer and its derivatives according to claim 9, characterized in that the ultrasonic power is 50-300W and the time is 1-30min.
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