Nano manganese adjuvant as well as preparation method and application thereof

Abstract

The invention provides a nano manganese adjuvant as well as a preparation method and application thereof, and belongs to the technical field of medicaments. The manganese nanoparticles Mn NPs are obtained through biomineralization with bovine serum albumin; and Mn NPs and an OVA solution are mixed by using a vortex method to prepare the manganese nanoparticles Mn / OVA loaded with the OVA antigen. On the premise that a functional material is not introduced, manganese is prepared into the nano-particles through simple operation. The reaction process is mild and controllable; the required chemical reagent amount is small, and the cost is reduced; the process is environment-friendly; a functional material is not introduced, so that the function of the manganese adjuvant is more favorably explored. The manganese adjuvant nano-particles prepared by the preparation method disclosed by the invention are relatively small in particle size and uniform in morphology, and show excellent dispersity and stability.

Description

Technical Field

[0001] The invention provides a nano manganese adjuvant and a preparation method and application thereof, belonging to the technical field of pharmaceuticals. Background Art

[0002] Vaccines are powerful means for specific infectious diseases. When they are applied to the human body, they will cause immune responses and establish specific immune memory. They are usually composed of two parts: antigens and adjuvants. Antigens are usually composed of proteins or carbohydrates from pathogens, which induce specific immune responses. However, the immunogenicity of current vaccines is weak and requires the help of adjuvants. Adjuvants are substances that stimulate and enhance the intensity and persistence of antigen immune responses, and can non-specifically enhance the body's immune response to vaccines. Therefore, one of the core strategies for vaccine development is to develop safe and effective new immune adjuvants. Aluminum salt adjuvants are the first adjuvants approved by the FDA for clinical application, but they can usually only induce classical humoral immunity and have weak activation ability for Th1 cell-mediated cellular immunity, which still limits the immune enhancement efficacy of aluminum adjuvants. At present, more and more studies are focusing on other types of metals (such as manganese and magnesium) to explore their immune enhancement effects in order to discover new adjuvants that are superior to metallic aluminum.

[0003] Manganese is a trace element necessary for a variety of biological activities, and many studies have reported that metallic manganese ions have adjuvant potential. In terms of mechanism, manganese can promote the production of cGAMP and enhance the binding affinity of cGAMP / STING to comprehensively promote cGAS-STING activation, thereby activating the host immune system, and is an important substance for the host to resist DNA viruses. Manganese can promote the uptake, presentation and formation of germinal centers of antigens by co-activating cGAS-STING signals and NLRP3 inflammasomes, thereby strongly enhancing cellular and humoral immune responses. At the same time, as a potential cGAS agonist, manganese ions can significantly increase the host's innate immune response to diseases such as Yersinia pseudotuberculosis infection. In addition, it has been reported that manganese can promote the maturation of dendritic cells and macrophages and the presentation of tumor-specific antigens, and enhance CD8 + Differentiation and activation of T cells and NK cells, and enhancement of CD8 + T cell memory has shown great potential in tumor immunotherapy, and combined with checkpoint inhibitors (anti-PD-1) has led to significant tumor regression. However, due to the high instability of manganese ions and their rapid metabolism in the body, their widespread application in the biomedical field is restricted.

[0004] Nanoparticles, as drug delivery carriers, can encapsulate and protect antigens. With their unique size effect and optimized surface properties, they can efficiently and accurately deliver antigens to key cells or lymphoid tissues in the immune system, thereby significantly enhancing the immune response. It has been reported that lipid nanoparticles efficiently deliver novel coronavirus mRNA antigens, protect antigens from degradation by nucleases, and promote mRNA to exert a strong specific immune response against the novel coronavirus. The vaccine has been successfully approved for marketing. In addition, due to their own foreignness and surface specificity, some nanoparticles such as PLGA nanoparticles and silica nanoparticles can induce an immune response in the body and have potential immune enhancement capabilities. This suggests that nano-manganese metal may significantly improve its stability and immunomodulatory effects after nano-crystallization, making it a potent adjuvant candidate.

[0005] For Mn 2+ There are problems such as instability and rapid metabolism in the body, which can easily lead to the inability to effectively exert the adjuvant effect. Many researchers have adopted different nanoformulation preparation methods such as hydration / solvothermal method, mixed coprecipitation method, solvent diffusion method, oil-in-water microemulsion method, thin film dispersion method, etc., and modified with different functional ligands (such as high molecular polymers, pectin polysaccharides, hyaluronic acid, etc.) to form a variety of functional manganese-containing nanoformulations (such as manganese dioxide nanoparticles / nanotubes / nano-warehouses, manganese-containing micelles, manganese-containing liposomes, etc.). However, the above preparation methods are mostly cumbersome to operate, difficult to remove inorganic solvents, poor biodegradability of the prepared materials, and the prepared manganese-containing nanoparticles have uneven particle size and instability. For example, manganese chloride (MnCl 2 ·4H 2 Manganese dioxide nanoparticles can be easily prepared by dissolving sodium oleate and sodium oleate in a mixed solution containing ethanol and n-hexane, and then stirring under high temperature and high pressure liquid environment. However, the preparation conditions are harsh and organic reagents are easy to remain. Pectin / MnO nanoparticles can be obtained by incubating manganese chloride solution (10%, w / v) with pectin microspheres prepared by emulsification-solvent diffusion method. 2+ However, the nanoparticles need to undergo repeated salt immersion centrifugation washing procedures, which can easily lead to uneven particle size and poor stability.

[0006] At the same time, the current research and application of manganese is mainly focused on anti-tumor immunity. The function of manganese in immunity is still largely unknown, and its mechanism of action is still unclear. The method of synthesizing manganese nanomaterials by introducing functional polymer compounds and inorganic substances will undoubtedly affect the exploration of manganese functions and mechanisms of action. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing antigen-carrying manganese adjuvant nanoparticles, which improves the stability of manganese nanoparticles while still exerting an immune-enhancing effect without affecting the exploration of manganese functions and mechanisms of action.

[0008] The specific technical solutions are:

[0009] A method for preparing a nano manganese adjuvant. Mainly includes the preparation of manganese nanoparticles (Mn NPs) and OVA antigen-loaded (Mn / OVANPs):

[0010] (1) Manganese nanoparticles (MnNPs) were prepared by biomineralization.

[0011] Prepare a certain concentration of raw material mother liquor:

[0012] Weigh 10 mg of bovine serum albumin (BSA) and dissolve it in 1 mL of distilled water to prepare a 10 mg / mL BSA solution, then weigh 12.6 mg of manganese chloride and dissolve it in 1 mL of distilled water to prepare a 12.6 mg / mL divalent manganese chloride mother solution. Both solutions are stored at 4°C.

[0013] Add 1 mL of distilled water to the vial, add different volumes of 10 mg / mL BSA solution to the vial and mix well, place the vial on a magnetic stirrer and stir at 37 °C, then slowly add 12.6 mg / mL of divalent manganese chloride mother solution to make BSA and divalent manganese chloride Mn 2+ The mass ratio W BSA / W Mn 2+ (8:1, 12:1, 16:1, 32:1), adjust the pH of the system in the vial to 11 with 1M NaOH solution, and continue stirring the reaction for 4 hours;

[0014] In the present invention, BSA and Mn 2+ The best mass ratio is 16:1, the best reaction conditions are stirring time of 4h and reaction temperature of 37℃.

[0015] After the reaction was completed, the reaction solution in the vial was transferred to a 30 kDa ultrafiltration tube and ultrafiltered twice at 5000 rpm and 4° C. for 25 min each time to remove excess NaOH solution and unreacted substrate.

[0016] Subsequently, the appropriate MnNPs production formula was screened and confirmed by changing the reaction time and temperature respectively.

[0017] (2) After confirming the preparation conditions of Mn NPs, different doses of Mn NPs were mixed with 1 mg / mL OVA solution by vortexing to prepare manganese nanoparticles Mn / OVA loaded with OVA antigen in different ratios.

[0018] Before vortex, according to Mn 2+ The mass ratio of W to OVA Mn 2+ / W OVA. Prepare different doses of MnNPs solution and OVA solution (0.5:1, 1:1, 2:1, 4:1, 8:1, 16:1), add MnNPs solution into the EP tube, add OVA solution while vortexing, vortex for 30 seconds, let stand for 30 minutes, and obtain Mn / OVA nanoparticles with different ratios.

[0019] The physicochemical properties of the manganese nanoparticles MnNPs and Mn / OVANPs obtained by the present invention mainly include the following aspects:

[0020] (1) Detection of MnNPs particle size, PDI and surface potential

[0021] The particle size and polydispersity coefficient of nanoparticles are important indicators for evaluating their preparation effect; surface potential refers to the potential of the cross-section between particles, also known as electromotive potential. This experiment uses dynamic light scattering to measure the particle size, PDI and surface potential of the prepared nanoparticle complex using a ZEN3600 laser particle size and potential analyzer. According to the operating steps of 1, after preparing Mn NPs, add them to a particle size dish or a potential dish, the measurement temperature is 20°C, the equilibrium time is 1 minute, and the appropriate number of cycles is set. Each sample is measured three times to measure the particle size, PDI and surface potential of the nanoparticles.

[0022] (2) Morphological observation of MnNPs

[0023] In addition to the dynamic light scattering method to measure the particle size distribution and potential of the nanoparticles, the morphology of the nanoparticles needs to be observed using a transmission electron microscope (TEM) to further ensure the successful preparation of the nanoparticles. Mn NPs were prepared according to method 1. The copper mesh covered with the support film was clamped with tweezers and placed on a round filter paper. The Mn NPs solution was then slowly dripped onto the copper mesh with a dropper until the manganese nanoparticle solution completely infiltrated the copper mesh. After natural drying for 5 minutes, the copper mesh was carefully picked up with tweezers, stained with 2% phosphotungstic acid for 20 seconds, and dried naturally. The morphological structure of the MnNPs was observed under TEM and photographed.

[0024] (3) Encapsulation efficiency and drug loading of MnNPs-loaded model antigens

[0025] The encapsulation efficiency of the model antigen OVA by Mn NPs was detected by ultrafiltration. Mn NPs encapsulating OVA were obtained according to the operation steps of 1. The OVA used in the experiment was RhB-OVA labeled with rhodamine B. The MnNPs encapsulating OVA were retained by an ultrafiltration tube (30 kDa). The liquid obtained after ultrafiltration in the outer tube was collected. RhB-OVA of different concentrations was configured as the standard well, and the liquid collected in the outer tube was used as the detection well. The fluorescence intensity was measured by an enzyme marker. The mass of the encapsulated OVA can be inferred from the fluorescence intensity of the solution in the standard well and the detection well and the concentration of the solution in the standard well. The encapsulation efficiency (EE) and drug loading rate of the nanoparticles were calculated using the following formula (1) and formula (2):

[0026]

[0027]

[0028] (4) Stability of MnNPs within 30 days

[0029] MnNPs were prepared according to the screening method in reference 1 and stored at 4°C. Dynamic light scattering technology was used to measure the changes in particle size, PDI and other data of the nanoparticle complex at different times on days 0, 1, 3, 5, 7, 9, 12, 15 and 30 to investigate its stability.

[0030] The in vitro cell experiments of Mn / OVANPs obtained by the present invention mainly include the following experiments:

[0031] (1) Cytotoxicity assay

[0032] When the confluence of cells in the culture dish reached more than 85%, 1.5×10 4 The number of cells was inoculated in a 96-well plate and cultured overnight. The next day, freshly prepared nanoparticles of different proportions were added and placed in a cell culture incubator for 24 hours. Then, a pre-prepared 10% CCK8 solution was added to each well in the dark and placed in a 37°C, 5% CO 2 After incubation for 0.5 to 4 hours under the conditions (the specific time can be adjusted according to the number, density and type of cells); when the color of the solution in the well plate turns orange, the absorbance at 450nm is measured with an enzyme marker. Set up 3 to 5 replicate wells for each group. The cell wells in the group without drug addition are used as the blank control group, which is regarded as 100% survival rate, and the wells without cell addition are used as the zero adjustment group, and the cell survival rate is calculated according to formula (3).

[0033] Cell survival rate (%) = (administered group - zero adjustment group) / (blank control group - zero adjustment group) × 100% (3)

[0034] (2) Cellular uptake experiment

[0035] When the confluence of cells in the culture dish reached more than 85%, 2×10 5 The number of cells was seeded in a 24-well plate and cultured overnight. The next day, freshly prepared Mn NP / OVA labeled with FITC and rhodamine B in different ratios were added and incubated for 1 hour. Subsequently, the cells were digested with trypsin and transferred to a 1.5mL EP tube, and then washed 2-3 times with PBS, all in the dark. Finally, the cells were resuspended with PBS, and the cell uptake efficiency on APCs was detected by flow cytometry.

[0036] The in vivo animal experiments of Mn / OVANPs obtained by the present invention are mainly as follows:

[0037] C57BL / 6 mice aged 4-6 weeks were randomly divided into 6 groups: Control, OVA, Algel 2% / OVA, Mn 2+ / OVA, L-Mn / OVA, M-Mn / OVA, H-Mn / OVA; 3-5 mice per group, the corresponding preparations were injected subcutaneously in the soles of the mice, and the injection volume of the preparation for each mouse was 25μL. The first day of injection was recorded as day 0, and the mice were injected for the second time on the 14th day to enhance the immune effect. The injection method and dose were consistent with the first injection. The mice were killed on the 28th day, and their blood, spleen and lymph nodes were taken for detection. The blood supernatant of the mice was collected by eyeball or orbital bleeding on the 14th and 28th days of mouse immunization, and the OVA-specific antibody IgG in the supernatant was detected by ELISA. In addition, the spleen was taken and ground, and the mouse spleen cell suspension was collected by lysis with red blood cell lysis buffer. The resulting spleen cell suspension was added to a 24-well plate, stimulated with OVA or SIINFEKL polypeptide, and 1000x brefeidinA was added, and incubated at 37℃ for 5h. Then add the flow cytometry fluorescent antibody of CD11c, CD80, CD8 or CD4, and incubate at 4°C in the dark for 30-60 minutes. For intracellular cytokine staining IFN-γ, fix and perforate with 4% paraformaldehyde and membrane permeabilization solution, then add the flow cytometry fluorescent antibody, and stain at room temperature in the dark for 20-30 minutes. After washing with PBS, resuspend the cells with an appropriate amount of PBS and detect by flow cytometry.

[0038] The present invention has the following technical effects:

[0039] 1. The present invention prepares manganese into nanoparticles through simple operations without introducing functional materials. The preparation process is concise and easy to operate; the reaction process is mild and controllable without harsh conditions;

[0040] 2. The method provided by the present invention is cost-effective and requires less chemical reagents, which greatly reduces costs;

[0041] 3. The process of the present invention is environmentally friendly and conforms to the development trend of green chemistry;

[0042] 4. The present invention does not introduce functional materials, which is more conducive to exploring the function of manganese adjuvant.

[0043] 5. The manganese adjuvant nanoparticles prepared by the present invention have small particle size, uniform morphology, and exhibit excellent dispersibility and stability.

[0044] 6. The nano manganese adjuvant obtained by the present invention can improve the uptake of antigens by antigen-presenting cells, induce safe and effective cellular immunity and humoral immune responses, and its immune effect is better than that of commercial aluminum gel adjuvants and manganese ions, which preliminarily verifies that manganese nanoparticles have the potential to be used as preventive vaccine adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The morphology of manganese nanoparticles H-Mn / OVA was observed by TEM in the example;

[0046] Figure 2 is the encapsulation efficiency of MnNPs in the examples, and the error bars represent the mean ± SEM;

[0047] Figure 3 is the drug loading of MnNPs in the examples, and the error bars represent the mean ± SEM;

[0048] Figure 4 The stability of the Mn NPs solution in the example within 30 days, the error bars represent the mean ± SEM;

[0049] Figure 5 The toxicity of MnNPs to RAW264.7 cells in the example;

[0050] Figure 6 The toxicity of MnNPs to DC2.4 cells in the example;

[0051] Figure 7 The toxicity of MnNPs to BMDCs cells in the embodiment;

[0052] Figure 8 The uptake of different doses of FITC-labeled BSA MnNPs / OVA in RAW264.7 cells in the example;

[0053] Fig. 9 The uptake of different doses of Mn NPs / OVA labeled with rhodamine B in RAW264.7 cells in the example;

[0054] Fig.10 The uptake of different doses of FITC-labeled BSA MnNPs / OVA in DC2.4 cells in the example;

[0055] Fig.11 The uptake of different doses of MnNPs / OVA labeled with rhodamine B in DC2.4 cells in the example;

[0056] Fig.12 The uptake of different doses of FITC-labeled BSA MnNPs / OVA in BMDCs cells in the example;

[0057] Fig.13 The uptake of different doses of Mn NPs / OVA labeled with rhodamine B in BMDCs cells in the example;

[0058] Fig.14 is the titer of specific antibody IgG in the antibody serum after the first immunization of the embodiment;

[0059] Fig.15 is the titer of specific antibody IgG in the antibody serum after the second immunization in the embodiment;

[0060] Fig.16 CD80 in lymph nodes after immunization in the example + CD11c + The situation of DC cells;

[0061] Fig.17 IFN-γ+CD8 in spleen cells of immunized mice + T cell count;

[0062] Fig.18 IFN-γ in spleen cells of immunized mice + CD4 + T cell count;

[0063] Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. DETAILED DESCRIPTION

[0064] The specific technical solution of the present invention is explained in conjunction with embodiments.

[0065] A method for preparing a nano manganese adjuvant. Mainly includes the preparation of manganese nanoparticles (Mn NPs) and OVA antigen-loaded (Mn / OVANPs):

[0066] (1) Manganese nanoparticles (Mn NPs) were prepared by biomineralization. In the early stage, a certain concentration of raw material mother liquor was prepared. 10 mg of bovine serum albumin (BSA) was weighed and dissolved in 1 mL of distilled water to prepare a 10 mg / mL BSA solution. Then 12.6 mg of manganese chloride was weighed and dissolved in 1 mL of distilled water to prepare a 12.6 mg / mL divalent manganese chloride mother liquor. Both solutions were stored at 4°C. 1 mL of distilled water was added to the vial, and different volumes of 10 mg / mL BSA solution were added to the vial to mix well. The vial was placed on a magnetic stirrer and stirred at 37°C. Then 12.6 mg / mL of divalent manganese chloride mother liquor was slowly added to allow BSA and divalent manganese chloride Mn to react. 2+ The mass ratio W BSA / W Mn 2+ (8:1, 12:1, 16:1, 32:1), adjust the pH of the system in the vial to 11 with 1M NaOH solution, and continue stirring the reaction for 4 hours; after the reaction, transfer the reaction solution in the vial to a 30kDa ultrafiltration tube, and ultrafilter twice at 5000rpm and 4℃ for 25 minutes each time to remove excess NaOH solution and unreacted substrate. Subsequently, the appropriate Mn NPs production formula was screened and confirmed by changing the reaction time and temperature.

[0067] (2) After confirming the preparation conditions of Mn NPs, different doses of Mn NPs were mixed with 1 mg / mL OVA solution by vortexing to prepare manganese nanoparticles Mn / OVA loaded with OVA antigen in different ratios. 2+ The mass ratio of W to OVA Mn 2+ / W OVA. Prepare different doses of Mn NPs solution and OVA solution (0.5:1, 1:1, 2:1, 4:1, 8:1, 16:1), add Mn NPs solution into the EP tube, add OVA solution while vortexing, vortex for 30 seconds, and let stand for 30 minutes to obtain Mn / OVA nanoparticles with different ratios (L-Mn / OVA, M-Mn / OVA, H-Mn / OVA, HH-Mn / OVA, HHH-Mn / OVA, HHHH-Mn / OVA).

[0068] 1. MnNPs prescription screening results and physical and chemical properties testing

[0069] In the screening process of MnNPs prescription, the present invention adjusts the BSA and Mn 2+ The mass ratio W BSA / W Mn 2+The nanoparticle formulations were screened by adjusting the ratio of BSA to Mn (8:1, 12:1, 16:1, 32:1), stirring time and reaction temperature. As shown in Table 1, Table 2 and Table 3, 2+ The mass ratio W BSA / W Mn 2+ When the ratio is 16:1, the stirring time is 4h and the reaction temperature is 37℃, the particle size and PDI are most suitable, which are the optimal prescription conditions for MnNPs.

[0070] Table 1 Different W BSA / W Mn 2+ Prepared Mn NPs

[0071]

[0072] Table 2 Mn NPs prepared at different stirring times

[0073]

[0074] Table 3 Compositions of Mn NPs at different temperatures

[0075]

[0076] Table 4 Different W Mn 2+ / W OVA Composition of Mn / OVANPs

[0077]

[0078] After determining the formula of the empty Mn NPs, the present invention needs to further prepare uniform and stable Mn NPs loaded with the model antigen OVA. 2+ The mass ratio W Mn 2+ / W OVA , six different ratios (0.5:1, 1:1, 2:1, 4:1, 8:1, and 16:1) of Mn NPs loaded with the model antigen OVA were prepared, which were denoted as L-Mn / OVA, M-Mn / OVA, H-Mn / OVA, HH-Mn / OVA, HHH-Mn / OVA, and HHHH-Mn / OVA, respectively. Mn 2+ / W OVA The particle size potential can be seen in Table 4. From the results, it can be seen that Mn NPs can well encapsulate OVA antigen to form nanoparticles of about 50-80nm, in which OVA and Mn 2+When the mass ratio of Mn increases from 1:2 to 2:1, the particle size will increase slightly, but the PDI value will decrease. 2+ The PDI will increase with increasing dose.

[0079] The morphology and structure of manganese nanoparticles Mn / OVA were observed by transmission electron microscopy (TEM). Under the transmission electron microscope, it can be clearly seen that the manganese nanoparticles are spherical in shape and have a relatively uniform particle size. Figure 1 , the scale in the lower left corner shows that the particle size of the nanoparticles is less than 100nm.

[0080] The present invention uses an ultrafiltration method to centrifuge the RhB-OVA that fails to bind to the Mn NPs and collect it in the outer tube of the ultrafiltration tube, and finally the average encapsulation rates of L-Mn / OVA, M-Mn / OVA and H-Mn / OVA are 98.43±0.47%, 97.98±0.11% and 99.06±0.53%, respectively; the average drug loadings are 0.61±0.003%, 0.31±0.001% and 0.16±0.001%, respectively. Figure 2 and Figure 3 .

[0081] The MnNPs solution was stored in a 4°C refrigerator and the particle size and polydispersity of the manganese nanoparticles were measured regularly within 30 days. Figure 4 The results showed that the particle size and polydispersity of manganese nanoparticles did not change significantly compared with day 0 within 30 days, proving that the nanoparticles had good stability.

[0082] 2. In vitro cell experiments of Mn / OVANPs

[0083] 2.1 Cytotoxicity test results of MnNPs

[0084] The safe and effective dosage range of manganese nanoparticles can prevent the situation where the dosage is too low and the manganese adjuvant has no effect or the dosage is too high and causes cell death. Mn 2+ / W OVA To investigate the toxicity of Mn / OVA at different mass ratios (dose ratios) on cells. Figure 5 , Figure 6 and Figure 7 It was observed that after 24 h of Mn / OVA administration, the mass ratio of manganese ions to OVA was Mn 2+ / W OVA When the ratio is less than or equal to 2:1, there is no difference between the cell survival rate of the drug-treated group and the cell survival rate of the non-drug-treated group. Mn 2+ / W OVA When the ratio was 4:1, there was no difference in the cell survival rate between the drug-treated group and the non-drug-treated group in DC2.4 cells, but the drug-treated group was toxic and damaged to the cells in RAW264.7 cells and BMDCs cells, with the cell survival rates of ~84.37% (**P<0.01) and ~69.67% (***P<0.001), respectively. This result shows that different cells have different tolerance to Mn / OVA. Mn 2+ / W OVA When the ratio is less than 2:1, the nanoformulation has no obvious toxicity to the three cells, which meets the basic characteristics of a delivery carrier. Mn 2+ / W OVA When the ratio is 4:1, the nanoparticle preparation has certain toxicity, while W Mn 2+ / W OVA Greater than 4:1 indicates that the nanoformulation has obvious toxicity. Based on the above results, in order to obtain a relatively safe, low-toxic and effective dosage of manganese nanoformulation, the present invention preliminarily selects the mass ratio of manganese ions to OVA W Mn 2+ / W OVA The ratios of 0.5:1, 1:1 and 2:1 (i.e. L-Mn / OVA, M-Mn / OVA, H-Mn / OVA) were used for subsequent cell experiments and animal immune effect studies.

[0085] Cellular uptake efficiency of 2.2Mn / OVANPs

[0086] In order for the adjuvant to exert its immune enhancement effect, it must first be taken up by antigen presenting cells, such as macrophages and dendritic cells. Therefore, the present invention prepares fluorescently labeled OVA-encapsulated manganese nanoparticles Mn / OVA by labeling BSA with FITC and OVA with rhodamine B, and detects the cell uptake effect of manganese nanoparticles by flow cytometry. Figures 8 to 13 As shown, compared with the Control or OVA group, different dose ratios of L-Mn / OVA, M-Mn / OVA, H-Mn / OVA and HH-Mn / OVA (W Mn 2+ / W OVAThe uptake efficiency of nanoparticles in RAW264.7 macrophages, DC2.4 dendritic cells and BMDCs cells was significantly increased with the increase of Mn dose. In addition, the uptake efficiency of H-Mn / OVA group was the highest in RAW264.7 cells, which was increased by 35 times and 93 times compared with OVA group and Control group, respectively (***P<0.001), and increased by 2 times compared with L-Mn / OVA and M-Mn / OVA (**P<0.01), but the uptake efficiency of HH-Mn / OVA decreased by 0.5 times compared with H-Mn / OVA (**P<0.01), which may be because the manganese ion content in HH-Mn / OVA is high and toxic to RAW264.7 cells. In summary, different doses of Mn / OVA can be effectively taken into cells.

[0087] 3. In vivo animal experiments of Mn / OVANPs

[0088] 3.1Mn / OVANPs can induce potent antibody response

[0089] Antibodies are a type of immunoglobulin that can specifically bind to antigens. They are important weapons for the body to protect against foreign substances such as bacteria and viruses. They are secreted by effector B cells and are mainly present in body fluids such as serum. They are important effector molecules that mediate humoral immunity. Therefore, antibody titer is used as an evaluation index for the humoral immune effect of manganese nanoadjuvants. According to the immunization method mentioned above, mice were immunized respectively, and the serum after two immunizations was taken for antibody content detection. Fig.14 and Fig.15 As shown in the figure, the antibody titer in the mouse serum was low after the first immunization. After the second immunization, H-Mn / OVA could induce strong IgG antibody titer, with an average titer of 5.25, which was significantly higher than Algel 2% / OVA and Mn 2+ / OVA, L-Mn / OVA and M-Mn / OVA groups, which shows that high-dose manganese nanoparticles H-Mn / OVA have a stronger adjuvant effect in enhancing humoral immune response.

[0090] 3.2Mn / OVANPs can effectively activate antigen presenting cells

[0091] To explore whether manganese nanoparticles have the ability to induce the maturation of peripheral lymph node cells, the axillary and inguinal lymph nodes on the same side as the injection site were collected for digestion, and the effect of manganese nanoparticles on activating the maturation of lymph node antigen-presenting cells was evaluated by detecting the number of dendritic cells expressing the co-stimulatory factor CD80 in the lymph nodes.

[0092] like Fig.16As shown in Figure 2, H-Mn / OVA significantly promoted the maturation of antigen-presenting cells in OVA-stimulated lymph nodes, and its CD80 + CD11c + The number of dendritic cells in the control group and OVA group was 3 times that of the control group and OVA group, and was also 3 times that of the M-Mn / OVA, Algel 2% / OVA and Mn 2+ / OVA group, indicating that the H-Mn / OVA group has the strongest effect in activating antigen-presenting cells. 2+ The effect of L-Mn / OVA in activating the maturation of antigen presenting cells in nearby lymph nodes in mice was far less than that of the manganese nanoparticle group, and the CD80 + CD11c + The number of dendritic cells is Mn 2+ / OVA group. Although the proportion of mature antigen-presenting cells in the lymph nodes of the L-Mn / OVA group was not much different from that of the H-Mn / OVA group, the effect varied greatly between individuals. In summary, H-Mn / OVA nanoparticles can effectively activate APCs, which is more effective than commercial aluminum adjuvant Algel 2% and free Mn 2+ Adjuvants are more competitive.

[0093] 3.3Mn / OVANPs can effectively induce CD8 + T cell response

[0094] CD8 + T cells are an important cell type in cellular immunity. In addition to investigating the enhancing effect of manganese nanoparticles on humoral immunity, this paper also explored its effect on cellular immunity, such as Fig.17 As shown in Figure 2, H-Mn / OVA nanoparticles can promote the production of IFN-γ in the spleen of OVA-stimulated mice. + CD8 + T cells, whose number is significantly higher than that of Mn 2+ / OVA (**P<0.01), Algel 2% / OVA group (***P<0.001), indicating that H-Mn / OVA has a significantly stronger ability to enhance cellular immune response than manganese ions and commercial aluminum adjuvants. At the same time, the lower doses of L-Mn / OVA and M-Mn / OVA groups could not increase IFN-γ + CD8 + The number of T cells indicated that high-dose H-Mn / OVA nanoparticles had a strong and effective cellular immune enhancement effect.

[0095] 3.4Mn / OVANPs can effectively induce CD4 + Th1 cell response

[0096] Th1 helper T cells are usually CD4 +When T cells mature, they will secrete IFN-γ, promote more Th1 differentiation, and participate in cellular immune response. Therefore, the present invention detects the number of Th1 cells that can secrete IFN-γ in the spleen of mice immunized with different preparations. Fig.18 As shown, after immunization with H-Mn / OVA nanoparticles, IFN-γ in spleen cells + CD4 + The number of Th1 cells was significantly higher than that of the other groups, increasing by 4.5 times compared with the OVA group (**P<0.01) and by 1.5 times compared with the Mn group. 2+ / OVA and Algel2% / OVA increased by ~2 times (*P<0.05), increased by 8.32 times compared with the M-Mn / OVA group (***P<0.001), and increased by 0.86 times compared with the L-Mn / OVA group (*P<0.05). The results showed that compared with other doses of Mn / OVA, H-Mn / OVA nanoparticles can effectively induce Th1 cell response, and are far superior to Mn 2+ and commercial aluminum adjuvant (Algel 2%).

[0097] In summary, the present invention adopts a simple preparation method to obtain manganese nanoparticles (Mn NPs) with uniform and stable particle size, which can safely and effectively deliver OVA into antigen-presenting cells, produce potent humoral immunity and cellular immunity enhancement effects, among which high-dose H-Mn / OVA has the strongest adjuvant effect.

Claims

1. A method for preparing a nano manganese adjuvant, characterized in that: The following steps are involved: (1) Preparation of manganese nanoparticles (MnNPs) by biomineralization; Prepare the raw material mother solution, dissolve bovine serum albumin BSA in distilled water to prepare BSA solution, and dissolve manganese chloride in distilled water to prepare divalent manganese chloride mother solution; both solutions are stored at 4°C; Add distilled water to the vial, add different volumes of BSA solution to the vial and mix well, place the vial on a magnetic stirrer for stirring, then slowly add divalent manganese chloride mother solution, adjust the pH of the system in the vial to 11 with 1M NaOH solution, and continue stirring the reaction; After the reaction is completed, the reaction solution in the vial is transferred to an ultrafiltration tube to remove excess NaOH solution and unreacted substrate by ultrafiltration; The appropriate MnNPs production formula was screened and confirmed by changing the reaction time and temperature respectively; (2) Preparation of OVA antigen-loaded Mn / OVANPs; After confirming the preparation conditions of MnNPs, MnNPs were mixed with OVA solution by vortexing to prepare manganese nanoparticles Mn / OVA loaded with OVA antigen in different ratios; Before vortex, according to Mn 2+ The mass ratio of W to OVA Mn 2+ / W OVA Prepare different doses of MnNPs solution and OVA solution, add MnNPs solution into the EP tube, add OVA solution while vortexing, vortex for 30 seconds, let stand for 30 minutes, and obtain Mn / OVA nanoparticles.

2. The method for preparing a nano manganese adjuvant according to claim 1, characterized in that: In step (1), a 10 mg / mL BSA solution is prepared, and a 12.6 mg / mL divalent manganese chloride mother solution is prepared.

3. The method for preparing a nano manganese adjuvant according to claim 1, characterized in that: In step (1), the BSA in the vial and the divalent manganese chloride Mn 2+ The mass ratio W BSA / W Mn 2+ It is 8 to 32:

1.

4. The method for preparing a nano manganese adjuvant according to claim 1, characterized in that: In step (1), the BSA in the vial and the divalent manganese chloride Mn 2+ The mass ratio W BSA / W Mn 2+ It is 16:

1.

5. The method for preparing a nano manganese adjuvant according to claim 1, characterized in that: The reaction conditions of the BSA solution and the divalent manganese chloride mother liquor in step (1) are: stirring and reacting at 37° C. for 4 hours.

6. The method for preparing a nano manganese adjuvant according to claim 1, characterized in that: Step (2) vortex Mn 2 + The mass ratio of W to OVA Mn 2+ / W OVA. It is 0.5 to 16:

1.

7. A nano manganese adjuvant obtained according to the preparation method according to any one of claims 1 to 6.

8. The nano manganese adjuvant according to claim 7, used as an adjuvant for preparing a vaccine.

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