Preparation method of nano material for antigen loading

Abstract

The invention belongs to the technical field of preparation of vaccine active component nano loading materials, and relates to a preparation method of a nano material for antigen loading. The method comprises the following steps: mixing a zinc chloride solution and a potassium ferricyanide solution, uniformly stirring, standing for reaction, centrifugally cleaning, and drying to obtain a Prussian blue analogue precipitate; and blending and tending the Prussian blue analogue precipitate and a polyethyleneimine solution, and then centrifuging to obtain the zinc-iron Prussian blue analogue / polyethyleneimine nano material. According to the invention, an inorganic nano-material is used as a carrier to load an antigen, and the used zinc-iron prussian blue analogue nano-material has a favorable effect on immune reaction. And Zn < 2 + > dissociated from the nano material can cause degradation of the mutated P53 gene, and a cGAS-STING pathway is recovered, so that the immune response is enhanced. And moreover, the immune response is further enhanced, the problem of autoimmunodeficiency is improved, and the clinical requirements on immunotherapy are more easily met.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of nanometer loading materials for active components of vaccines, and relates to a method for preparing a nanometer material for antigen loading. Background Art

[0002] Dendritic cells (DCs) are an important component of the immune system and have attracted widespread attention in the field of biomedical research since they were first discovered in 1973. DC cells are a powerful professional antigen-presenting cell that can efficiently take up, process and present antigens. They are one of the most effective ways to activate the body's immune system and stimulate resistance to cancer invasion. Based on these characteristics of DC cells, the study of DC activation in vivo and in vitro has gradually become a frontier field in cancer immunotherapy and vaccine development.

[0003] At present, the most important issue in DC activation is how to efficiently utilize antigens (such as OVA), amplify immune responses, and make up for the problem of insufficient autoimmunity. With the continuous development of nanomaterials, the use of nanomaterials as antigens or drug carriers to improve the effect of immunotherapy has received widespread attention. In addition, the development of immune signaling pathways also has an important impact on immunotherapy research. The cGAS-STING signaling pathway has become an important innate immune pathway that affects almost all aspects of tumorigenesis. Simply put, cGAS recognizes cytoplasmic DNA molecules and catalyzes STING to produce type i interferon, activating innate immunity. Metal ions, especially Mn 2+ and Zn 2+ ions, which can effectively make the cGAS-STING pathway sensitive to cytoplasmic DNA. Therefore, the use of nanomaterials that are beneficial to the immune signaling pathway as carriers of antigenic live drugs is of great significance for improving the immune effect. Nano vaccines have attracted more and more attention due to their various advantages over traditional vaccines and their potential to improve the effectiveness of vaccination. The use of nanocarriers to deliver antigens has the advantages of protecting the loaded antigens from degradation, reducing the antigen dose, promoting the tumor vaccination cascade, prolonging the vaccination effect, and shaping the immune type. With the continuous advancement of nanotechnology and the deepening of research, such nanocarriers have been more widely used in the development and research of immune vaccines. By optimizing the design of nanomaterials, improving their safety and effectiveness, and deeply exploring the mechanism of their interaction with the immune system, it is expected that more efficient and safe vaccine carriers will be developed, providing better options for the prevention of infectious diseases and immunotherapy of cancer. Summary of the invention

[0004] Aiming at the problems existing in the use of traditional antigen active components, the present invention proposes a novel method for preparing nanomaterials for antigen loading.

[0005] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: A method for preparing a nanomaterial for antigen loading, comprising the following steps: (1) Mix a zinc chloride solution and a potassium ferrocyanide solution to obtain a mixed solution A, stir the mixed solution evenly and then allow the mixed solution to react. After the reaction is completed, centrifuge the mixed solution. Wash the solid obtained by centrifugation and dry it to obtain a Prussian blue analog precipitate.

[0006] (2) The Prussian blue analog precipitate is mixed and incubated with the polyethyleneimine solution, and then centrifuged to obtain the zinc-iron Prussian blue analog / polyethyleneimine nanomaterial.

[0007] Preferably, in step (1), the concentrations of the zinc chloride solution and the potassium ferrocyanide solution are both 5-20 mM, and the volume ratio of the zinc chloride solution to the potassium ferrocyanide solution is 1:1; the static reaction temperature is 15-40° C., and the static reaction time is 12-48 h.

[0008] Preferably, in step (2), the mass ratio of the Prussian blue analog precipitate to the polyethyleneimine solution is 1:4-6, and the incubation time is 40-100 h.

[0009] The method for preparing the nanomaterial loaded with OVA by the above method comprises the following steps: adding zinc-iron Prussian blue analogue / polyethyleneimine nanomaterial into deionized water to obtain a dispersion, adding OVA into the dispersion to make its concentration reach 20-700 μg / ml, incubating at 4°C for 10-20 hours; and centrifuging to obtain zinc-iron Prussian blue analogue / polyethyleneimine / OVA.

[0010] Polyethyleneimine is a cationic polymer, and the amine group can be protonated at neutral pH, so that the surface of the nanoparticles is positively charged, which makes it easier to adsorb antigens, and the amino groups on the surface of polyethyleneimine are combined with the carboxyl groups on the surface of the protein, which is conducive to the subsequent OVA grafting. Prussian blue analogs have good biocompatibility, low toxicity in cell experiments, and by adjusting the size of nanomaterials, cells can more easily internalize materials and produce immune responses. Polyethyleneimine is a polymer with cationic properties of nitrogen atoms, so it can show positive charges in solution, which is conducive to reacting with negatively charged substances, and the Zeta potential characterization of Prussian blue analogs shows that the material is negatively charged under non-strong acid conditions, so polyethyleneimine can be loaded on the surface of zinc iron Prussian blue through electrostatic action. The present invention uses zinc iron Prussian blue analog nanomaterials as carriers to activate DCs to produce a series of immune responses, which makes up for the shortcomings of autoimmune responses and achieves rapid and efficient immune responses. During the synthesis of zinc iron Prussian blue analogue / polyethyleneimine / OVA, the synthesis time of zinc iron Prussian blue analogue, the loading amount of polyethyleneimine and the grafting amount of OVA need to be strictly controlled. Controlling the synthesis time of zinc iron Prussian blue analogue means controlling the crystal size of zinc iron Prussian blue analogue, ensuring that the inorganic material is synthesized to be less than 300nm for easy cellular endocytosis.

[0011] Compared with the prior art, the advantages and positive effects of the present invention are: The present invention uses inorganic nanomaterials as carriers to load antigens, and the zinc-iron Prussian blue analog nanomaterials used have a beneficial effect on immune response. 2+ It can lead to the degradation of the mutated P53 gene and restore the cGAS-STING pathway, thereby enhancing the immune response. Loading OVA antigen is more conducive to DCs forming antibodies and then activating downstream immune responses. The combination of the two materials not only has good biocompatibility but also further enhances the immune response, improves the problem of autoimmune deficiency, and is more likely to meet the clinical requirements for immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a scanning electron micrograph of the zinc-iron Prussian blue analog synthesized by chemical precipitation in Example 1.

[0013] Figure 2 This is a graph showing the cytotoxicity of DC cells stained with CCK-8 and co-cultured with different concentrations of zinc-iron Prussian blue analogs in Example 1.

[0014] Figure 3 The comparison results of OVA loading rate before and after modification of the material with polyethyleneimine in Example 1 are shown.

[0015] Figure 4The results of flow cytometry of CD86+ content of cells co-cultured with materials of different concentrations in Example 1 are shown. DETAILED DESCRIPTION

[0016] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0017] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0018] Example 1 (1) Prepare 10 mL of 10 mM zinc chloride solution and 10 mL of 10 mM potassium ferrocyanide solution. Ultrasonicate the two solutions for 10 min and then mix them. Stir them manually with a glass rod for 2 min to mix them evenly. Then let them stand naturally for 12 h to obtain a zinc-iron Prussian blue analog nanomaterial suspension. Centrifuge at 8000 r / min for 5 min. Wash the solid obtained by centrifugation with deionized water three times and dry it in an oven at 60°C for 5 h to obtain a zinc-iron Prussian blue analog for later use.

[0019] (2) The zinc iron Prussian blue analog was dispersed in deionized water to obtain a dispersion with a concentration of 1 mg / mL. Then, 1 mL of the dispersion was added to 1 mL of a 5 mg / mL polyethyleneimine solution. The reaction was stirred at 35°C for 64 h at a stirring rate of 200 rpm. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min. The solid obtained by centrifugation was rinsed three times with deionized water to obtain zinc iron Prussian blue analog / polyethyleneimine nanoparticles.

[0020] (3) Disperse the zinc iron Prussian blue analog / polyethyleneimine nanoparticles in deionized water to obtain a dispersion with a concentration of 1 mg / mL, and add OVA (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥98%) into deionized water to prepare an OVA solution with a concentration of 50 μg / mL. Take 1 mL of the dispersion, add 1 mL of the OVA solution, and then incubate naturally at 4°C for 12 hours, centrifuge at 13000 rpm for 10 minutes, and obtain the supernatant and the lower layer of nanoparticles loaded with OVA.

[0021] The OVA concentration in the supernatant was detected using the BCA protein detection kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) according to the operating instructions to determine the OVA loading amount.

[0022] The electron microscopic image of the zinc iron Prussian blue analogue obtained in step (1) is as follows: Figure 1 ,from Figure 1 It can be seen that the size of the nanoparticles is below 300 nanometers.

[0023] To investigate the toxicity of the nanomaterials prepared in step (1), a 96-well plate was used, and 100 μL of 2×10 5 BMDC cell culture medium (45mL 1640 culture medium + 5mL serum + 500 μL double antibody, the same below. 1640 culture medium was purchased from Thermo Fisher Scientific, serum was purchased from Corning Life Sciences Co., Ltd., and double antibody was purchased from Stefan Biotechnology Co., Ltd.), and cultured for 24 hours to allow the cells to settle to the bottom of the well plate. Then, the materials prepared in step (1) were added to different well plates so that the concentration of the materials in each well plate was 0, 10, 20, 40, and 80 μg / mL, and five replicate wells were made for each concentration. The materials and cells were co-cultured at 37°C and 5% CO2 for 24 hours, the well plates were removed and centrifuged, the supernatant was poured out, and then a 10% mass fraction of cck-8 cell dye (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) diluted with PBS solution (Lanjieke Technology Co., Ltd., each small package was dissolved in 1L of deionized water, the same below) was added to each well plate, and the staining lasted for more than half an hour. The cell survival degree was tested using an enzyme marker. The results are as follows Figure 2 Compared with the control group with a concentration of 0, the cell activity did not decrease significantly at other concentrations, and the reason for the slight increase was that the cells proliferated a small amount. Therefore, the synthesized zinc-iron Prussian blue analog nanomaterial has good biocompatibility.

[0024] Figure 3 The results of the OVA loading rate test of the nanomaterial (ZnFe-PEI) prepared in this example are compared with the material (ZnFe) that was not incubated with the polyethyleneimine solution. Figure 3 As shown, under the same conditions, the material modified with polyethyleneimine has a higher loading rate.

[0025] Detection of the effects of OVA-loaded nanoparticles on DC cells.

[0026] 6-8 week old c57 mice (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.) were selected and the mice were devitalized by cervical dislocation. The hair and muscle tissue of the mice were cut off with scissors, and the mice were washed twice with 75% alcohol and PBS solution respectively, and the leg bones were removed for use. The leg bones of the mice were ground in a mortar in a clean bench, and the primary BMDC cells were filtered using a 100μm cell strainer (purchased from Changde Beekman Biotechnology Co., Ltd.), and the strainer was rinsed with culture medium. The collected filtrate was centrifuged at 1000r / min for 5min, the supernatant was poured out, and the cells were resuspended in 500μL of BMDC cell culture medium. 10mL of BMDC culture medium was added to the culture dish, and the resuspended cells were added to the culture dish. After that, 10mL of BMDC culture medium was added to the culture dish on the third day, and half of the medium in the culture dish was replaced on the sixth day (half of the volume of culture medium was removed and an equal amount of fresh BMDC culture medium was added), and half of the medium in the culture dish was replaced on the eighth day, and the sample could be collected on the tenth day.

[0027] The cultured cells were spread on 12-well plates, with 1 mL of BMDC culture medium per well. A total of 10 wells were used, with 2 × 10 cells per well. 6 The 12-well plates were placed in static culture for 24 hours, and then the OVA-loaded nanoparticles were added to the cell solution according to the concentration gradient (the concentration gradient was 0, 10, 20, 40, 80 μg / mL). Two replicate wells were set for each concentration gradient, and the materials and cells were co-cultured for 24 hours under normal cell culture conditions. The cultured cell mixture was transferred to a flow tube, and 0.5 μL of antibody (purchased from Biolegend Biotechnology Co., Ltd.) was added to each tube for 30 minutes of reaction, and then washed twice with PBS solution (1000 r / min, 5 minutes), and 200 μL of paraformaldehyde (purchased from Lanjieke Technology Co., Ltd.) was added to each well for fixation, and then the content of related cytokines was examined by flow cytometry. Figure 4 The results of flow cytometry show that as the concentration of the material increases, CD86 + The content of CD86 + The results showed that OVA-loaded nanoparticles themselves had a significant stimulatory effect on cells.

[0028] Example 2 This embodiment provides a method for preparing a nanomaterial for antigen loading, and the steps are as follows. The parts not specifically described in this embodiment are consistent with those in Example 1.

[0029] (1) Preparation of zinc-iron Prussian blue analog nanomaterials Prepare solution A with 20 mM zinc chloride and solution B with 20 mM potassium ferrocyanide. Ultrasonicate solution A and solution B for 10 min to dissolve them evenly and then mix them. Stir them evenly and let them react for 12 hours to obtain a zinc-iron Prussian blue analog solution. Centrifuge at 8000 r / min and wash with deionized water for 3 times to obtain a zinc-iron Prussian blue analog precipitate. Dry at 60°C for later use.

[0030] (2) Preparation of zinc iron Prussian blue analog @polyethyleneimine @OVA material 1 mL of zinc iron Prussian blue analog (1 mg / mL) was dispersed in 1 mL of polyethyleneimine solution (5 mg / mL) and stirred at 35°C for 64 h. The product was centrifuged and washed with water three times to obtain zinc iron Prussian blue analog @ polyethyleneimine. The nanoparticles (1 mg / mL) were mixed with 100 μg / mL of OVA solution. After incubation at 4°C for 12 h, the supernatant was collected and centrifuged at 13000 rpm for 10 min. The OVA concentration in the supernatant was detected using the BCA protein detection kit according to the manufacturer's protocol to determine the OVA loading amount.

[0031] Example 3 This embodiment provides a method for preparing a nanomaterial for antigen loading, and the steps are as follows. The parts not specifically described in this embodiment are consistent with those in Example 1.

[0032] (1) Preparation of zinc-iron Prussian blue analog nanomaterials Prepare solution A with 10 mM zinc chloride and solution B with 10 mM potassium ferrocyanide, dissolve solutions A and B evenly by ultrasonication for 10 min, mix them, stir evenly and let them stand for 24 h to obtain a zinc-iron Prussian blue analog solution, centrifuge at 8000 r / min and wash with deionized water for 3 times to obtain a zinc-iron Prussian blue analog precipitate, and dry at 60°C for later use; (2) Preparation of zinc iron Prussian blue analog @polyethyleneimine @OVA material 1 mL of zinc iron Prussian blue analog (1 mg / mL) was dispersed in 1 mL of polyethyleneimine solution (5 mg / mL) and stirred at 35°C for 64 h. The product was centrifuged and washed with water three times to obtain zinc iron Prussian blue analog @ polyethyleneimine. The nanoparticles (1 mg / mL) were mixed with 100 μg / mL of OVA solution. After incubation at 4°C for 20 h, the supernatant was collected and centrifuged at 13000 rpm for 10 min. The OVA concentration in the supernatant was detected using the BCA protein detection kit according to the manufacturer's protocol to determine the OVA loading amount.

[0033] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a nanomaterial for antigen loading, characterized in that: Here are the steps: (1) mixing a zinc chloride solution and a potassium ferrocyanide solution to obtain a mixed solution A, stirring the mixed solution evenly and then allowing the mixed solution to react. After the reaction is completed, centrifuging the mixed solution, washing the solid obtained by centrifugation and drying the solid to obtain a Prussian blue analog precipitate; (2) The Prussian blue analog precipitate is mixed and incubated with the polyethyleneimine solution, and then centrifuged to obtain the zinc-iron Prussian blue analog / polyethyleneimine nanomaterial.

2. The method for preparing the antigen-loaded nanomaterial according to claim 1, characterized in that: In step (1), the concentrations of the zinc chloride solution and the potassium ferrocyanide solution are both 5-20 mM, and the volume ratio of the zinc chloride solution to the potassium ferrocyanide solution is 1:1; the static reaction temperature is 15-40° C., and the static reaction time is 12-48 h.

3. The method for preparing the antigen-loaded nanomaterial according to claim 1, characterized in that: In step (2), the mass ratio of the Prussian blue analog precipitate to polyethyleneimine is 1:4-6, and the incubation time is 40-100 hours.

4. A method for loading OVA on a nanomaterial prepared by any one of claims 1 to 3, characterized in that: The zinc-iron Prussian blue analog / polyethyleneimine nanomaterial is added into deionized water to obtain a dispersion, OVA is added into the dispersion to make its concentration reach 20-700 μg / ml, and the dispersion is incubated at 4°C for 10-20 hours; After centrifugation, zinc iron Prussian blue analog / polyethyleneimine / OVA was obtained.

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