Manganese dioxide nano adjuvant, preparation method and application thereof

Abstract

The application provides a manganese dioxide nanomaterial, which comprises manganese dioxide nanoparticles and a carrier substance combined on the surface of the manganese dioxide nanoparticles; the carrier substance comprises CpG oligonucleotide and / or antigen; the antigen comprises protein antigen and / or polypeptide antigen. The manganese dioxide nanomaterial provided by the application greatly improves the immune stimulation of the immune adjuvant CpG, is used for improving the immune protection function of a vaccine, and solves the inherent defects that the oligonucleotide CpG as an immune stimulant is difficult to enter the lymph nodes of the body, and a large amount of CpG is needed to effectively improve the immune response; and as a vaccine adjuvant, the manganese dioxide nanomaterial can carry antigen, especially protein antigen, effectively improves the uptake of immune cells in the lymph nodes to the antigen, and thus improves the immunogenicity of the antigen. Meanwhile, the nanomaterial is biodegradable, and in a weak acid environment such as a lysosome, the nanomaterial can be gradually degraded into manganese ions (Mn 2+ ), and finally discharged out of the body.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biological medicine and vaccine technology, and particularly relates to a manganese dioxide nanomaterial and a preparation method and application thereof, in particular to a manganese dioxide nano-adjuvant and a preparation method and application thereof. BACKGROUND

[0002] A vaccine is an immunological preparation for preventing infectious diseases, which is made of pathogenic microorganisms (such as bacteria, rickettsia, viruses, etc.) and their metabolites by artificial attenuation, inactivation or genetic engineering methods. The vaccine retains the characteristics of stimulating the body's immune system. When the animal body contacts the pathogen without harm, the immune system will produce certain protective substances, such as cytokines, active physiological substances, and special antibodies. When the body contacts the pathogen again, the immune system will follow its original memory to produce more protective substances to prevent the harm of the pathogen. Since its inception, vaccines have played a huge role in protecting human health. However, vaccines for some major diseases, especially infectious diseases such as AIDS, malaria, and malignant tumors, have not yet been developed. Vaccines are mainly composed of antigens (mostly protein antigens or polypeptides) and immune adjuvants. Therefore, effectively improving the immunogenicity of antigens and the immune activation of adjuvants are two main strategies for vaccine development.

[0003] At present, due to its unique physicochemical properties, nanomaterials have shown good application prospects as carriers in improving the immunogenicity of antigens. So far, most of the nanomaterials mainly carry antigens by wrapping, covalent connection or electrostatic adsorption, etc. to promote the uptake of antigens by immune cells. However, the antigen carrying capacity of these materials is not ideal, and it is difficult to effectively improve the immunogenicity of antigens.

[0004] As the only clinically approved vaccine adjuvant, aluminum adjuvant can effectively promote the induction of humoral immune response by antigens, but it is difficult to induce cellular immune response, which is obviously not suitable for eliminating endogenous pathogens such as HIV and tumor cells. At the same time, aluminum adjuvant has certain side effects, which can cause inflammation and allergic reactions at the injection site.

[0005] Therefore, how to develop a more suitable and safer new type of vaccine adjuvant has become a key strategy in the field of vaccine development, and it is also one of the focuses of many forward-looking researchers in the field. SUMMARY

[0006] Therefore, the present application aims to provide a manganese dioxide nanomaterial, a preparation method and application thereof, in particular, a manganese dioxide nano-adjuvant, a preparation method and application thereof.

[0007] The present application provides a manganese dioxide nanomaterial, comprising manganese dioxide nanoparticles and a carrier substance complexed on the surface of the manganese dioxide nanoparticles.

[0008] The carrier substance comprises CpG oligonucleotides and / or antigens.

[0009] The antigens comprise protein antigens and / or polypeptide antigens.

[0010] Preferably, the particle size of the manganese dioxide nanomaterial is 15-100 nm.

[0011] Preferably, the molar ratio of the manganese dioxide to the CpG oligonucleotides is 0.8:(0.006-0.02).

[0012] Preferably, the molar ratio of the manganese dioxide to the protein antigens is 0.8:(0.02-0.06).

[0013] The molar ratio of the manganese dioxide to the polypeptide antigens is 0.8:(0.06-0.2).

[0014] Preferably, the antigens comprise one or more of HIV antigens, Mycobacterium tuberculosis antigens, malaria antigens, human papilloma virus antigens and tumor-related antigens.

[0015] The present application provides a preparation method of a manganese dioxide nanomaterial, comprising the following steps:

[0016] A) mixing a manganese chloride solution and a carrier substance solution, and then incubating to obtain a complex;

[0017] The carrier substance comprises CpG oligonucleotides and / or antigens; the antigens comprise protein antigens and / or polypeptide antigens.

[0018] B) reacting the complex obtained in the above step with a sodium hydroxide solution to obtain a manganese dioxide nanomaterial.

[0019] Preferably, the molar ratio of the manganese chloride to the CpG oligonucleotides is 1:(0.006-0.02).

[0020] The molar ratio of the manganese chloride to the protein antigens is 1:(0.02-0.06).

[0021] The molar ratio of the manganese chloride to the polypeptide antigen is 1:(0.06-0.2);

[0022] The molar ratio of the manganese chloride to the sodium hydroxide is 1:(8-32).

[0023] Preferably, the concentration of the manganese chloride solution is 80-200 μg / mL;

[0024] The concentration of the CpG oligonucleotide solution is 20-60 μg / mL;

[0025] The concentration of the protein antigen solution is 650-1500 μg / mL;

[0026] The concentration of the polypeptide antigen solution is 90-200 μg / mL;

[0027] The concentration of the sodium hydroxide solution is 300-800 μg / mL.

[0028] Preferably, the incubation time is 15-30 minutes;

[0029] The reaction time is 1-3 hours.

[0030] The manganese dioxide nanomaterial of any one of the above technical solutions or the manganese dioxide nanomaterial prepared by any one of the above technical solutions is applied in the field of vaccines.

[0031] The present application provides a manganese dioxide nanomaterial, which comprises manganese dioxide nanoparticles and a carrier substance complexed on the surface of the manganese dioxide nanoparticles; the carrier substance comprises CpG oligonucleotides and / or antigens; the antigens comprise protein antigens and / or polypeptide antigens. Compared with the prior art, the existing vaccine adjuvant is difficult to induce a cellular immune response, has the disadvantage of being unable to clear endogenous pathogens, and the aluminum adjuvant also has certain side effects, such as causing inflammation and allergic reactions at the injection site. The manganese dioxide nanomaterial provided by the present application greatly improves the immune stimulating effect of the immune adjuvant CpG, so that it can be used to improve the immune protection function of the vaccine, effectively solving the inherent defects that the oligonucleotide CpG as an immune stimulant is difficult to enter the lymph nodes of the body, and a large amount of CpG needs to be used to effectively improve the immune response; and as a vaccine adjuvant, it can effectively carry antigens, especially protein antigens or polypeptide antigens, effectively improve the uptake of immune cells in the lymph nodes to the antigens, and greatly improve the immunogenicity of the antigens. At the same time, the nanoadjuvant is biodegradable, and in a weak acid environment such as a lysosome, it can gradually degrade into manganese ions (Mn 2+ ), and finally be discharged out of the body.

[0032] The experimental results show that compared with free CpG and chicken ovalbumin antigen, the manganese dioxide nano-adjuvant (MnO2-CpG, MnO2-OVA) can more effectively activate professional antigen-presenting cells-dendritic cells and the mass secretion of related cytokines (interleukin 12-IL-12 and tumor necrosis factor-TNF-α); the MnO2-CpG nano-adjuvant can greatly promote the cross-presentation efficiency of protein antigen, thereby greatly benefiting the activation of the cellular immune response of the body; the animal experiment results further show that without the help of other fluorescent molecules, the magnetic resonance imaging function of the manganese dioxide nano-vaccine (MnO2-CpG+MnO2-OVA nano-adjuvant mixture) can provide direct information for determining the optimal immune cycle, the nano-adjuvant vaccine can greatly improve the cross-presentation efficiency of the antigen by promoting the effective migration of OVA antigen and CpG immune activator to the lymph node, thereby greatly promoting the occurrence of the antigen-specific cellular immune response of the body, including greatly enhancing the killing function and proliferation ability of CD8 + T lymphocytes (CTL) and the mass production of interferon (IFN-γ) of the body, and finally effectively inhibiting the malignant progression of the tumor. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Transmission electron microscope photos of the MnO2-OVA nanoparticles and the MnO2-CpG nanoparticles prepared in Example 1 of the present application;

[0034] Figure 2 Dynamic light scattering characterization curve diagrams of the MnO2-OVA nanoparticle solution and the MnO2-CpG nanoparticle solution obtained in Example 2 of the present application;

[0035] Figure 3 Acid responsiveness and magnetic resonance imaging evaluation results of the MnO2-OVA nanoparticle solution and the MnO2-CpG nanoparticle solution obtained in Example 2 of the present application;

[0036] Figure 4 Cytotoxicity evaluation results of the MnO2-OVA nanoparticles and the MnO2-CpG nanoparticles obtained in Example 3 of the present application on dendritic cells;

[0037] Figure 5 Evaluation results of the influence of the MnO2-OVA nanoparticles and the MnO2-CpG nanoparticles in Example 4 on the maturation of dendritic cells and the cross-presentation efficiency of antigens;

[0038] Figure 6 Evaluation results of the influence of the manganese dioxide nano-adjuvant vaccine (MnO2-OVA+MnO2-CpG) in Example 5 on the cellular immune response of mice and the in vivo level cross-presentation efficiency of antigens;

[0039] Figure 7 The results of the evaluation of the preventive effect of the manganese dioxide nano adjuvant vaccine (MnO2-OVA+MnO2-CpG) in Example 6 of the present application on melanoma stably expressing OVA antigen;

[0040] Figure 8 A schematic diagram of the synthesis of the manganese dioxide nanomaterial provided by the present application is shown. DETAILED DESCRIPTION

[0041] In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application and are not a limitation on the patent claims of the present application.

[0042] All raw materials of the present application are not particularly limited in source, and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0043] All raw materials of the present application are not particularly limited in purity, and the present application preferably uses conventional purity in the field of biochemical reagents, biological pharmaceutical technology or vaccines.

[0044] The present application provides a manganese dioxide nanomaterial, which comprises manganese dioxide nanoparticles and a carrier substance complexed on the surface of the manganese dioxide nanoparticles;

[0045] The carrier substance comprises CpG oligonucleotides and / or antigens;

[0046] The antigens comprise protein antigens and / or polypeptide antigens.

[0047] The definition of the CpG oligonucleotides of the present application is not particularly limited, and the CpG oligonucleotides (CpG-ODN) well known to those skilled in the art can be used, i.e., oligonucleotides containing CPG motifs, which can be adjusted and selected by those skilled in the art according to actual application conditions, product requirements and functional uses.

[0048] The content of the CpG oligonucleotides of the present application is not particularly limited, and the conventional loading content well known to those skilled in the art can be used, which can be adjusted and selected by those skilled in the art according to actual application conditions, product requirements and functional uses, and the molar ratio of the manganese dioxide to the CpG oligonucleotides of the present application is preferably 0.8:(0.006-0.02), more preferably 0.8:(0.007-0.014), more preferably 0.8:(0.009-0.012), and specifically can be 0.8:0.009.

[0049] The antigen is not particularly limited in the present application, and can be a conventional antigen known to those skilled in the art, which can be adjusted and selected by those skilled in the art according to the actual application, product requirements and functional use. The antigen in the present application preferably includes a protein antigen and / or a polypeptide antigen, and more preferably a protein antigen or a polypeptide antigen.

[0050] The specific selection of the antigen in the present application is not particularly limited, and can be a conventional antigen known to those skilled in the art, which can be adjusted and selected by those skilled in the art according to the actual application, product requirements and functional use. The antigen in the present application preferably includes one or more of HIV antigens, Mycobacterium tuberculosis antigens, malaria antigens, human papilloma virus antigens and tumor-associated antigens. In the present application, specifically, chicken egg white albumin is used as a model antigen for substitution research on the above-mentioned antigens in the research stage.

[0051] The content of the antigen in the present application is not particularly limited, and can be a conventional carrier content known to those skilled in the art, which can be adjusted and selected by those skilled in the art according to the actual application, product requirements and functional use. The molar ratio of MnO2 to the protein antigen in the present application is preferably 0.8:(0.02-0.06), more preferably 0.8:(0.025-0.055), more preferably 0.8:(0.03-0.05), and specifically can be 0.8:0.05. The molar ratio of MnO2 to the polypeptide antigen is preferably 0.8:(0.06-0.2), more preferably 0.8:(0.09-0.15), more preferably 0.8:(0.1-0.13), and specifically can be 0.8:0.1.

[0052] The particle size of the MnO2 nanomaterial in the present application is not particularly limited, and can be a conventional particle size of a nanomaterial known to those skilled in the art, which can be adjusted and selected by those skilled in the art according to the actual application, product requirements and functional use. The particle size of the MnO2 nanomaterial in the present application is preferably 15-100 nm, more preferably 18-80 nm, more preferably 20-60 nm, and more preferably 23-40 nm, and the average particle size can be 25 nm.

[0053] The specific method of the complex in the present application is not particularly limited, and can be a carrier and a carried substance known to those skilled in the art, which can be adjusted and selected by those skilled in the art according to the actual application, product requirements and functional use. The complex in the present application preferably includes one or more of adsorption, grafting and coordination.

[0054] In the formation process of the MnO2 nanoparticles in the present application, first, Mn 2+The coordination with the carboxyl or sulfydryl in the CpG oligonucleotide or protein or polypeptide antigen is followed by the formation of the MnO2 particles under alkaline conditions. The CpG oligonucleotide and / or the protein antigen or polypeptide antigen is located on the surface of the MnO2 particles, and can play the role of dispersant by electrostatic repulsion due to the negative electric property, thereby inhibiting the agglomeration of the particles, so as to obtain the manganese dioxide nanoparticles.

[0055] The application further provides a preparation method of the manganese dioxide nanomaterial, comprising the following steps:

[0056] A) mixing the manganese chloride solution and the supported substance solution, and then incubating (i.e. standing at room temperature) to obtain a complex;

[0057] The supported substance comprises the CpG oligonucleotide and / or the antigen; the antigen comprises the protein antigen and / or the polypeptide antigen;

[0058] B) reacting the complex obtained in the above step with a sodium hydroxide solution to obtain the manganese dioxide nanomaterial.

[0059] The properties, structures and proportions of the raw materials or products in the preparation method are consistent with the preferred principles and specific preferred schemes of the manganese dioxide nanomaterial, and will not be repeated here.

[0060] The application first mixes the manganese chloride solution and the supported substance solution, and then incubates to obtain a complex; the supported substance comprises the CpG oligonucleotide and / or the antigen; the supported substance comprises the CpG oligonucleotide and / or the antigen; the antigen comprises the protein antigen and / or the polypeptide antigen.

[0061] In the application, the supported substance can be the CpG oligonucleotide and the antigen. When two different supported substances are added at the same time, the manganese dioxide nanomaterial simultaneously loaded with the CpG oligonucleotide and the antigen can be formed, or the manganese dioxide nanomaterial loaded with the CpG oligonucleotide and the antigen respectively can be formed. The person skilled in the art can select and adjust according to the actual needs, product requirements and quality requirements. Moreover, the manganese dioxide-CpG nanoadjuvant (the manganese dioxide loaded with the CpG oligonucleotide) can also be mixed with the manganese dioxide-antigen nanoadjuvant (the manganese dioxide loaded with the antigen) for use as a vaccine to induce effective immune protection.

[0062] The concentration of the manganese chloride solution is not particularly limited, and the concentration of the conventional manganese chloride solution known to the person skilled in the art can be used. The person skilled in the art can adjust and select according to the actual production situation, product requirements and functional use. The concentration of the manganese chloride solution is preferably 80-200 μg / mL, more preferably 100-180 μg / mL, and more preferably 120-160 μg / mL.

[0063] The concentration of the carried substance solution is not particularly limited, and a conventional concentration known to those skilled in the art can be used. Those skilled in the art can adjust and select the concentration of the carried substance solution, i.e., the concentration of the CpG oligonucleotide solution, according to actual production conditions, product requirements, and functional uses. The concentration of the CpG oligonucleotide solution is preferably 20-60 μg / mL, more preferably 30-50 μg / mL, and more preferably 35-45 μg / mL. The concentration of the protein antigen solution is preferably 650-1500 μg / mL, more preferably 750-1400 μg / mL, more preferably 850-1300 μg / mL, and more preferably 950-1200 μg / mL. The concentration of the polypeptide antigen solution is preferably 90-200 μg / mL, more preferably 110-180 μg / mL, more preferably 130-160 μg / mL, and more preferably 140-150 μg / mL.

[0064] The amount of the CpG oligonucleotide added is not particularly limited, and a conventional amount known to those skilled in the art can be used. Those skilled in the art can adjust and select the amount of the CpG oligonucleotide added according to actual application conditions, product requirements, and functional uses. The molar ratio of the manganese chloride to the CpG oligonucleotide in the present application is preferably 1:(0.006-0.02), more preferably 1:(0.007-0.014), and more preferably 1:(0.009-0.012), and specifically can be 1:0.009. In the present application, due to objective factors in practice, the molar content of manganese in the product is equivalent to about 80% of the amount of manganese added.

[0065] The amount of the antigen added is not particularly limited, and a conventional amount known to those skilled in the art can be used. Those skilled in the art can adjust and select the amount of the antigen added according to actual application conditions, product requirements, and functional uses. The molar ratio of the manganese dioxide to the protein antigen in the present application is preferably 1:(0.02-0.06), more preferably 1:(0.025-0.055), and more preferably 1:(0.03-0.05), and specifically can be 1:0.05. The molar ratio of the manganese dioxide to the polypeptide antigen is preferably 1:(0.06-0.2), more preferably 1:(0.09-0.15), and more preferably 1:(0.1-0.13), and specifically can be 1:0.1. In the present application, due to objective factors in practice, the molar content of manganese in the product is equivalent to about 80% of the amount of manganese added.

[0066] The mixing method is not particularly limited, and the mixing method known to those skilled in the art can be used, and those skilled in the art can adjust and select the mixing method according to the actual production situation, product requirements and functional use.

[0067] In the process of magnetic stirring, the loaded substance solution (CpG oligonucleotide solution and / or antigen solution) is added dropwise into the manganese chloride solution.

[0068] The speed of the dropwise addition is not particularly limited, and the speed of the dropwise addition known to those skilled in the art can be used, and those skilled in the art can adjust and select the speed of the dropwise addition according to the actual production situation, product requirements and functional use.

[0069] The specific time of the incubation is not particularly limited, and the time known to those skilled in the art can be used, and those skilled in the art can adjust and select the time according to the actual production situation, product requirements and functional use, and the time of the incubation is preferably 15-30 minutes, more preferably 18-28 minutes, and more preferably 20-25 minutes.

[0070] In the present application, in the above-mentioned mixing and incubation process, the manganese chloride and the CpG or the protein antigen or the polypeptide antigen are combined together to form a complex through biomineralization, i.e. Mn 2+ coordinates with the carboxyl or sulfhydryl in the CpG or the protein or the polypeptide, and combines together to form a complex.

[0071] The complex obtained in the above-mentioned step is then reacted with a sodium hydroxide solution to obtain a manganese dioxide nanomaterial.

[0072] The amount of the sodium hydroxide is not particularly limited, and the amount known to those skilled in the art can be used, and those skilled in the art can adjust and select the amount according to the actual application situation, product requirements and functional use, and the molar ratio of the manganese chloride to the sodium hydroxide is preferably 1:(8-32), more preferably 1:(11.5-25), and more preferably 1:(12-20).

[0073] In the present application, when the carried substance is CpG oligonucleotide, the molar ratio of manganese chloride to sodium hydroxide is more preferably 1:(8-12), more preferably 1:(8-10), and specifically can be 1:8; when the carried substance is protein antigen, the molar ratio of manganese chloride to sodium hydroxide is more preferably 1:(10-30), more preferably 1:(12-25), more preferably 1:(15-22), and specifically can be 1:20; when the carried substance is polypeptide antigen, the molar ratio of manganese chloride to sodium hydroxide is more preferably 1:(8-15), more preferably 1:(9-14), more preferably 1:(10-12), and specifically can be 1:10.

[0074] The present application does not have a particular limitation on the specific time of the reaction, and the conventional reaction time known to those skilled in the art can be used, and those skilled in the art can adjust and select according to the actual production situation, product requirements and functional use. The reaction time of the present application is preferably 1-3 hours, and more preferably 1.5-2.5 hours.

[0075] The present application adds sodium hydroxide to induce the following chemical reaction, see formula (I)

[0076] 2MnCl2+4NaOH+O2→MnO2+4NaCl+4H2O (I).

[0077] In the process of forming MnO2, CpG and / or antigen are located on the surface of MnO2 particles, and due to their negative charge, they can play a dispersing role and inhibit particle agglomeration, ultimately obtaining manganese dioxide nanoparticles.

[0078] The present application does not have a particular limitation on the temperature of the preparation method, and the conventional temperature known to those skilled in the art can be used, and those skilled in the art can adjust and select according to the actual production situation, product requirements and functional use. The temperature of the preparation method of the present application is preferably room temperature, i.e. 15-25℃.

[0079] In order to ensure the performance of the product, the overall preparation process is optimized, and the reaction preferably further includes a post-treatment step. The present application does not have a particular limitation on the post-treatment step, and the post-treatment step known to those skilled in the art can be used, and those skilled in the art can adjust and select according to the actual production situation, product requirements and functional use. The post-treatment step of the present application preferably includes a separation step, and more specifically preferably dialysis.

[0080] The present application also provides the application of the manganese dioxide nanomaterial of any one of the above technical solutions or the manganese dioxide nanomaterial prepared by any one of the above technical solutions in the field of vaccines.

[0081] The application has no particular restriction on the specific aspects of the application, and the skilled person in the art can adjust and select according to the actual situation, product requirements and functional use, and the application of the application includes a vaccine nano adjuvant, which can be a nano adjuvant for enhancing the activity of an immune adjuvant or the immunogenicity of an antigen, and the manganese dioxide-CpG nano adjuvant can also be used in combination with the manganese dioxide-antigen nano adjuvant, as a vaccine to induce effective immune protection.

[0082] The above steps of the application provide a manganese dioxide nanomaterial and a preparation method and application thereof, the application carries CpG or antigen through biomimetic mineralization, the synthesis method is simple, suitable for large-scale production and application, has large carrying capacity, can effectively promote the immunogenicity of protein antigens or polypeptide antigens and the immune stimulating activity of oligonucleotide CpG as a nano adjuvant. Moreover, it is acid-responsive and biodegradable, when the nano adjuvant is taken into immune cells such as dendritic cells, it can be gradually degraded into manganese ions in the acidic environment of lysosomes, and finally discharged out of the body; at the same time, it can also change the integrity of the lysosome membrane during the degradation process, effectively improving the cross-presentation efficiency of antigens; in addition, the manganese dioxide nano adjuvant prepared by the application also has the function of magnetic resonance imaging, which can provide important guidance for optimizing the immune strategy.

[0083] Reference Figure 8 , Figure 8 The application provides a schematic diagram of the synthesis of the manganese dioxide nanomaterial.

[0084] The experimental results show that, compared with free CpG and chicken ovalbumin antigen, the manganese dioxide nano adjuvant (MnO2-CpG, MnO2-OVA) can more effectively activate professional antigen-presenting cells-dendritic cells and the massive secretion of related cytokines (interleukin 12-IL-12 and tumor necrosis factor-TNF-α); the MnO2-CpG nano adjuvant can also greatly promote the cross-presentation efficiency of protein antigens, thereby greatly facilitating the activation of the cellular immune response of the body; the animal experiment results further show that, without the help of other fluorescent molecules, the magnetic resonance imaging function of the manganese dioxide nano vaccine (MnO2-CpG+MnO2-OVA nano adjuvant mixture) can provide direct information for determining the optimal immune cycle, the nano adjuvant vaccine can effectively migrate to the lymph nodes by promoting the migration of OVA antigen and CpG immune activator, greatly improving the cross-presentation efficiency of antigens, thereby greatly promoting the occurrence of antigen-specific cellular immune response of the body, including greatly enhancing the killing function and proliferation ability of CD8 + T lymphocytes (CTL) and the massive production of interferon (IFN-γ) of the body, and finally effectively inhibiting the malignant progression of tumors.

[0085] In order to further illustrate the present application, the following embodiments are used to describe the manganese dioxide nanomaterial and the preparation method and application thereof in detail. However, it should be understood that the embodiments are implemented on the premise of the technical scheme of the present application, and the detailed implementation mode and specific operation process are given, which are only used to further illustrate the features and advantages of the present application, and are not used to limit the present application, and the protection scope of the present application is not limited to the following embodiments.

[0086] Example 1

[0087] Preparation of manganese dioxide-antigen (MnO2-OVA) and manganese dioxide-CpG (MnO2-CpG) nano-adjuvant

[0088] Firstly, 0.5 mL of OVA solution (45 μM) or CpG solution (16 μM) is added dropwise into 1 mL of manganese chloride solution (MnCl2, 1 mM) under magnetic stirring, and incubated at room temperature for 15-30 minutes; then, 12 μL of sodium hydroxide solution (1 M) is added into the above solution, and the reaction is continued for 1.5 hours; finally, the reaction solution is placed in a dialysis bag (with a molecular weight cut-off of 3500 Da) for dialysis for 4-6 hours, and the fresh dialysis solution (deionized water) is replaced every hour, so that the yellow-brown MnO2-OVA and MnO2-CpG nano-adjuvant can be obtained.

[0089] The concentration of MnO2 in the MnO2-OVA and MnO2-CpG nano-adjuvant prepared in Example 1 of the present application is detected by inductively coupled plasma atomic emission spectrometry (ICP-AES), and the results show that the concentration of MnO2 in the two kinds of nano-adjuvant is 0.53 mM, that is, about 80% of MnCl2 is finally converted into MnO2.

[0090] The particle size and surface charge of the MnO2-OVA and MnO2-CpG nano-adjuvant prepared in Example 1 of the present application are characterized.

[0091] Referring to Figure 1 , Figure 1 The transmission electron microscope photos of the MnO2-OVA and MnO2-CpG nanoparticles prepared in Example 1 of the present application are shown.

[0092] Referring to Figure 2 , Figure 2 The dynamic light scattering characterization curve of the MnO2-OVA and MnO2-CpG nanoparticles prepared in Example 1 of the present application is shown.

[0093] From Figure 1 and Figure 2It can be seen that the particle size and potential of the MnO2-OVA and MnO2-CpG nanomaterials prepared in Example 1 of the present application are 37.4±4.6 nm and -20.5±2.0 mV, and 27.9±2.3 nm and -19.5±2.2 mV, respectively.

[0094] Example 2

[0095] Acid responsiveness and magnetic resonance imaging of manganese dioxide nanoadjuvant

[0096] The MnO2-OVA and MnO2-CpG nanoadjuvants were diluted 5 times with phosphate buffer solutions with different pH conditions (pH 7.4, pH 5.0) and incubated at room temperature, and the color change of the solution was observed. Meanwhile, the above four solutions were gradiently diluted, and then the T1 enhanced magnetic resonance signal (R1) was detected by a magnetic resonance imaging instrument.

[0097] Reference Figure 3 , Figure 3 The acid responsiveness and magnetic resonance imaging evaluation results of the MnO2-OVA nanoparticle solution and the MnO2-CpG nanoparticle solution obtained in Example 2 of the present application.

[0098] The results are shown in Figure 3 As shown in the results, the MnO2-OVA and MnO2-CpG changed from yellow-brown to colorless solution in an acidic environment, indicating that the manganese dioxide nanoadjuvant was dissolved into manganese ions. At the same time, the results showed that the MnO2-OVA and MnO2-CpG could significantly enhance the T1 magnetic resonance imaging signal in an acidic environment.

[0099] Example 3

[0100] Evaluation of cytotoxicity of manganese dioxide nanoadjuvant on dendritic cells

[0101] Firstly, the C57 mice were sacrificed by cervical dislocation, and the bone marrow was extracted aseptically. After lysing the red blood cells, a single cell suspension was prepared, and the cell count was performed. Then, the single cell suspension was diluted to 4×105 / mL with RPMI1640 complete medium (containing 10% fetal bovine serum, penicillin-streptomycin and β-mercaptoethanol) containing granulocyte-macrophage colony-stimulating factor (GM-CSF), inoculated into a bacterial culture dish (diameter 6 cm), and placed in a cell culture incubator for culture; on the 3rd day, the same volume of fresh medium (containing GM-CSF) was supplemented, and on the 6th day, half of the medium was replaced, and on the 7th day, 90% pure dendritic cells were obtained and reserved.

[0102] MnO2-OVA and MnO2-CpG were gradiently diluted with RPMI1640 medium without stimulating factors, mixed with appropriate amount of dendritic cells, inoculated into 96-well culture plates, and placed in a cell culture incubator for 24 hours, and then 3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl tetrazolium bromide (MTT) was used to detect cell viability.

[0103] Referring to Figure 4 , Figure 4 The results of the evaluation of the cytotoxicity of the MnO2-OVA nanoparticles and the MnO2-CpG nanoparticles on dendritic cells obtained in Example 3 of the present application are shown in Table 1.

[0104] The results are shown in Table 1. Figure 4 MnO2-OVA and MnO2-CpG are essentially non-toxic to dendritic cells, and even at the highest concentration, the cell viability remains above 90%.

[0105] Example 4

[0106] Evaluation of the effect of manganese dioxide nano-adjuvant on dendritic cell maturation and antigen cross-presentation efficiency

[0107] According to the results of the cell viability evaluation in Example 3, negative control group, positive control group (lipopolysaccharide-LPS), CpG, GpC, MnO2-CpG, OVA, OVA+CpG, OVA+MnO2-CpG, MnO2-OVA, MnO2-OVA+CpG, MnO2-OVA+MnO2-CpG were set up for incubation with dendritic cells for 24 hours, wherein the working concentrations of OVA, CpG and LPS were 20 μg / mL, 0.8 μg / mL and 1 μg / mL respectively, and the concentration of MnO2 was 16 μM.

[0108] Then, the changes in dendritic cell maturation (CD11c+CD80+CD86+) and OVA antigen cross-presentation (OVA257-264+CD11c+) efficiency were evaluated using a flow cytometer; at the same time, the expression levels of mature related cytokines IL-12p40 and tumor necrosis factor (TNF-α) in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).

[0109] Referring to Figure 5 , Figure 5 The results of the evaluation of the effect of the MnO2-OVA nanoparticles and the MnO2-CpG nanoparticles on dendritic cell maturation and antigen cross-presentation efficiency in Example 4 of the present application are shown in Table 2.

[0110] The results are shown in Table 2. Figure 5As shown in AC, compared with CpG, MnO2-CpG nanoadjuvant significantly promoted the maturation of dendritic cells and the secretion of related cytokines; compared with OVA, MnO2-OVA nanoadjuvant significantly promoted the secretion of IL-12p40 and TNF-α; and compared with other treatment groups, MnO2-OVA+MnO2-CpG had the strongest activation effect on dendritic cells. Simultaneously, the results indicated that MnO2-CpG significantly improved the cross-presentation efficiency of OVA antigen (…). Figure 5 D), which is extremely beneficial for inducing cellular immune responses.

[0111] Example 5

[0112] The effect of manganese dioxide nanoadjuvant vaccines on mouse cellular immune responses

[0113] Female C57BL / 6 mice were used in the experiment. Seven groups were established: PBS, OVA, OVA+CpG, OVA+MnO2-CpG, MnO2-OVA, MnO2-OVA+CpG, and MnO2-OVA+MnO2-CpG, with six mice in each group. Mice were immunized via intradermal injection, once every two weeks for a total of three immunizations. Two weeks after the third immunization, the mice were sacrificed. Figure 6 A) Splenocytes were aseptically collected from mice. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to detect changes in T cell secretion of interferon-gamma, CD8+ T lymphocyte cytotoxicity, CD8+ T lymphocyte proliferation, serum interferon-gamma expression levels, and immune response tendency. Simultaneously, three days after the first immunization, inguinal lymph nodes were aseptically collected, and flow cytometry was used to analyze changes in antigen cross-presentation (the ratio of OVA257-264+CD8+ T cells).

[0114] See Figure 6 , Figure 6 This is the evaluation result of the effect of the manganese dioxide nano-adjuvant vaccine (MnO2-OVA+MnO2-CpG) in Example 5 of the present invention on mouse cellular immune response and in vivo antigen cross-presentation efficiency.

[0115] The results are as follows Figure 6 As shown, compared with other immunization groups, the MnO2-OVA+MnO2-CpG nanoadjuvant vaccine significantly promoted CD8+ T lymphocyte-mediated cellular immune responses. Figure 6 BD), promotes the expression of γ-interferon in serum ( Figure 6 E), and it can significantly promote the occurrence of immune responses that predispose to cellular immunity in the body. Figure 6 F), these results are associated with their ability to significantly improve antigen cross-presentation efficiency at the in vivo level (F). Figure 6G) is closely related.

[0116] Example 6

[0117] Evaluation of the preventive effect of manganese dioxide nano-adjuvant vaccines against melanoma

[0118] Following the method described in Example 5, two weeks after the third immunization, mice were subcutaneously injected with 3×10⁵ melanoma cells (B16-OVA) or B16 melanoma cells stably expressing OVA antigen (immunoreactivity specificity test) to evaluate the preventive effect of manganese dioxide nanoadjuvant vaccine on melanoma cells.

[0119] See Figure 7 , Figure 7 This is the evaluation result of the preventive effect of the manganese dioxide nano-adjuvant vaccine (MnO2-OVA+MnO2-CpG) in Example 6 of the present invention on melanoma stably expressing OVA antigen.

[0120] The results are as follows Figure 7 As shown, the manganese dioxide nano-adjuvant vaccine (MnO2-OVA+MnO2-CpG) can specifically and effectively inhibit the growth of B16-OVA melanoma.

[0121] The foregoing has provided a detailed description of a manganese dioxide nano-adjuvant, its preparation method, and its applications. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these embodiments are merely for the purpose of helping to understand the method and core ideas of the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the wording of the claims, or if they include equivalent structural elements that are not substantially different from the wording of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A manganese dioxide nanomaterial, characterized in that, The application relates to a manganese dioxide nanomaterial, which comprises (1) manganese dioxide nanoparticles and a carrier substance complexed on the surface of the manganese dioxide nanoparticles, the carrier substance being a CpG oligonucleotide; and (2) manganese dioxide nanoparticles and a carrier substance complexed on the surface of the manganese dioxide nanoparticles, the carrier substance being an antigen, the antigen comprising a protein antigen and / or a polypeptide antigen; wherein the manganese dioxide nanoparticles are used as an immunoadjuvant to enhance an immune response.

2. The manganese dioxide nanomaterial of claim 1, wherein, The particle size of the manganese dioxide nanomaterial is 15-100 nm.

3. The manganese dioxide nanomaterial of claim 1, wherein, The molar ratio of the manganese dioxide to the CpG oligonucleotide is 0.8: (0.006-0.02).

4. The manganese dioxide nanomaterial of claim 1, wherein, The molar ratio of the manganese dioxide to the protein antigen is 0.8: (0.02-0.06). The molar ratio of the manganese dioxide to the polypeptide antigen is 0.8: (0.06-0.2).

5. The manganese dioxide nanomaterial of claim 1, wherein, The antigen is selected from one or more of an HIV antigen, a tubercle bacillus antigen, a malaria antigen and a tumor-related antigen.

6. The manganese dioxide nanomaterial of claim 1, wherein, The antigen is a human papilloma virus antigen.

7. A method for preparing the manganese dioxide nanomaterial of claim 1, characterized in that, The application further relates to a preparation method of the manganese dioxide nanomaterial, which comprises the following steps: A) mixing a manganese chloride solution and a CpG oligonucleotide solution, a manganese chloride solution and an antigen solution respectively, and then incubating to obtain a complex, the antigen comprising a protein antigen and / or a polypeptide antigen; B) reacting the complex obtained in the above step with a sodium hydroxide solution to obtain the manganese dioxide nanomaterial. 8.The method of claim 7, wherein the manganese dioxide nanomaterial is prepared by the method of claim 1, and the manganese dioxide nanomaterial has a particle size of 10-100 nm. The molar ratio of the manganese chloride to the CpG oligonucleotide is 1: (0.006-0.02). The molar ratio of the manganese chloride to the protein antigen is 1: (0.02-0.06). The molar ratio of the manganese chloride to the polypeptide antigen is 1: (0.06-0.2). The molar ratio of the manganese chloride to the sodium hydroxide is 1: (8-32). 9.The method of claim 7, wherein the manganese dioxide nanomaterial is prepared by the method of claim 1, and the manganese dioxide nanomaterial has a particle size of 10-100 nm. The concentration of the manganese chloride solution is 80-200 mu g / mL. The concentration of the CpG oligonucleotide solution is 20-60 mu g / mL. The concentration of the protein antigen solution is 650-1500 mu g / mL. The concentration of the polypeptide antigen solution is 90-200 mu g / mL. The concentration of the sodium hydroxide solution is 300-800 mu g / mL. 10.The method of claim 7, wherein the manganese dioxide nanomaterial is prepared by the method of claim 1, and the manganese dioxide nanomaterial is characterized in that, The incubation time is 15-30 minutes. The reaction time is 1-3 hours.

11. The manganese dioxide nanomaterial of any one of claims 1-6 or prepared by any one of claims 7-10 for use in the preparation of a vaccine.

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