Cationic manganese nano adjuvant as well as preparation method and application thereof

Abstract

The invention relates to a cationized manganese nano adjuvant as well as a preparation method and application thereof. The cationized manganese nano adjuvant comprises manganous-manganic oxide nano particles and cationized template molecules wrapping the surfaces of the manganous-manganic oxide nano particles, the cationization template molecule is a protein subjected to cationization modification treatment, or a protein fragment thereof, or a polypeptide fragment thereof. Precise regulation and control of surface charges and structures of the manganese nanoparticles are realized through a biomineralization technology, and the key problems of a traditional vaccine adjuvant in the aspects of antigen delivery, immune activation and biocompatibility are effectively solved. The cationized manganese nano adjuvant can efficiently adsorb various antigens to form a stable nano vaccine compound, and congenital immunity and adaptive immunity response of an organism can be remarkably activated; lymph nodes can be effectively targeted, the enrichment degree of antigens in the lymph nodes is improved, and the immune effect is further enhanced; meanwhile, the biotoxicity is low, and the stability is excellent.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology and relates to a cationic manganese nanoadjuvant, its preparation method, and its application. Background Technology

[0002] Vaccines are a key means of preventing and controlling infectious diseases, tumors, and other illnesses, and their effectiveness is closely related to the performance of the adjuvant. While traditional vaccine adjuvants have played a role to some extent, they have many limitations and urgently need improvement. Aluminum adjuvants are currently the most widely used vaccine adjuvants; they can promote an immune response to antigens to a certain extent, and their safety is widely recognized. However, aluminum adjuvants have significant drawbacks: they lack the ability to effectively activate cellular immunity, making it difficult to induce a comprehensive and efficient immune response. Therefore, in dealing with diseases that require cellular immunity, such as viral infections and tumors, they cannot fully exert the protective effect of vaccines.

[0003] Achieving efficient antigen delivery is a major challenge in vaccine development. Lymph nodes, as key organs of the immune system, are important sites for the aggregation of immune cells and the initiation of immune responses. However, due to the size limitations and unique structure of lymph nodes, accurately delivering vaccines to immune cells and enabling them to exert their effects is extremely difficult. One of the main problems currently facing subunit-based vaccines is the difficulty in achieving satisfactory antigen delivery, including targeting lymph nodes and ensuring effective cellular uptake, which limits the vaccine's immunizing efficacy.

[0004] With the development of nanotechnology and metalloimmunology, metal-based nanomaterials have shown potential application value in the vaccine field. Manganese participates in various physiological processes in organisms and has good biocompatibility, which lays the foundation for the development of manganese nanoadjuvants. However, manganese nanoadjuvants are negatively charged, and most natural antigen proteins are also negatively charged under physiological conditions, limiting their adsorption and loading efficiency for antigens. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a cationic manganese nanoadjuvant, its preparation method, and its application, specifically relating to a cationic manganese nanoadjuvant and its preparation method, and a nanovaccine and its preparation method.

[0006] To achieve this objective, the present invention employs the following technical solution:

[0007] In a first aspect, the present invention provides a cationic manganese nanoadjuvant, the cationic manganese nanoadjuvant comprising manganese tetroxide nanoparticles and a cationic template molecule coated on the surface of the manganese tetroxide nanoparticles, wherein the cationic template molecule is a protein, a protein fragment thereof, or a polypeptide fragment thereof that has undergone cationic modification.

[0008] This invention achieves precise control over the surface charge and structure of manganese nanoparticles through biomineralization technology, effectively solving key problems in antigen delivery, immune activation, and biocompatibility of traditional vaccine adjuvants. This cationic manganese nanoadjuvant can efficiently adsorb various antigens, forming stable nanovaccine complexes. For naturally negatively charged antigens, its adsorption rate is significantly higher than that of manganese nanoadjuvants. In terms of immune activation, it can significantly upregulate the expression of co-stimulatory molecules on the surface of dendritic cells and promote cytokine secretion, thereby activating the body's innate and adaptive immune responses. Simultaneously, it can effectively target lymph nodes, with higher targeting efficiency than free antigens, increasing antigen accumulation in lymph nodes and further enhancing the immune effect. Furthermore, it exhibits low biotoxicity and lower cytotoxicity than manganese ions. Moreover, it demonstrates excellent stability, with no significant change in particle size after 6 months of storage at 4°C. This adjuvant is suitable for antiviral vaccines, tumor vaccines, and other fields, providing a safe and efficient adjuvant option for the development of novel vaccines.

[0009] Preferably, the molar ratio of the cationized template molecule to manganese is 1:(10-1000), such as 1:10, 1:30, 1:50, 1:80, 1:100, 1:200, 1:300, 1:400, 1:500, 1:700, 1:1000, etc.

[0010] Preferably, the protein comprises any one or a combination of at least two of bovine serum albumin, human serum albumin, mouse serum albumin, transferrin, horse spleen ferritin, human recombinant heavy chain ferritin, or antigenic protein.

[0011] Preferably, the cationization modification treatment involves coupling the cationization modifier to the template molecule using a coupling agent.

[0012] Preferably, the cationizing modifier includes any one or a combination of at least two of ethylenediamine, propylenediamine, butyldiamine, pentanediamine, or hexamethylenediamine.

[0013] Preferably, the coupling agent comprises any one or a combination of at least two of the following: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-carbonyldiimidazole (CDI), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM), and benzotriazol-1-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP).

[0014] Preferably, the molar ratio of the template molecule to the coupling agent is 1:(1-3000), for example, 1:10, 1:20, 1:30, 1:40, 1:60, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:1000, 1:1500, 1:1800, 1:2000, 1:2300, 1:2500, 1:3000, etc.

[0015] Preferably, the molar ratio of the template molecule to the cationic modifier is 1:(1-3000), for example, 1:10, 1:20, 1:30, 1:40, 1:60, 1:80, 1:100, 1:150, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:1000, 1:1500, 1:1800, 1:2000, 1:2300, 1:2500, 1:3000, etc.

[0016] All other point values ​​not listed above within the range of the above values ​​can be selected, and will not be elaborated on here.

[0017] In a second aspect, the present invention provides a method for preparing the cationic manganese nanoadjuvant according to the first aspect, the preparation method comprising the following steps:

[0018] (1) Dissolve the template molecule in a buffer solution, mix it with a coupling agent and a cationizing agent, react it under stirring, and purify the product by dialysis or ultrafiltration to obtain the cationized template molecule.

[0019] (2) Mix the aqueous solution of divalent manganese salt with the cationic template molecule solution to obtain a composite solution; adjust the pH of the composite solution to alkaline, react under stirring, and purify the product by dialysis or ultrafiltration to obtain the cationic manganese nanoadjuvant.

[0020] The preparation method of the cationic manganese nanoadjuvant involved in this invention is achieved through a two-step process: first, a coupling agent is used to couple the cationic reagent to the template molecule, and then a biomineralization reaction under alkaline conditions is used to generate manganese nanoparticles with the surface coated with cationic template molecules.

[0021] Preferably, the concentration of the template molecule dissolved in the buffer solution in step (1) is 1-50 mg / mL, such as 10 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, etc.

[0022] Preferably, the reaction in step (1) is carried out at pH 2-7 (e.g., pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, etc.), the reaction temperature is 0-50℃ (e.g., 4℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 50℃, etc.), the reaction time is 0.5-12h (e.g., 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h, 12h, etc.), and the stirring speed is 200-1200rpm (e.g., 200rpm, 400rpm, 600rpm, 800rpm, 1000rpm, 1200rpm, etc.).

[0023] Preferably, the divalent manganese salt in step (2) includes any one or a combination of at least two of manganese chloride, manganese nitrate, manganese acetate, or manganese sulfate.

[0024] Preferably, the concentration of the divalent manganese salt aqueous solution is 0.1-1 mol / L, such as 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 1 mol / L, etc.

[0025] Preferably, the concentration of the cationized template molecule solution is 1-20 mg / mL, such as 1 mg / mL, 3 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, etc.

[0026] Preferably, the pH of the composite solution is adjusted to 9-12, such as pH=9, pH=9.5, pH=10, pH=10.5, pH=11, pH=11.5, pH=12, etc.

[0027] Preferably, the pH of the composite solution is adjusted by an alkaline reagent, which includes any one or a combination of at least two of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, triethylamine, pyridine, N-methylmorpholine, or tetramethylethylenediamine.

[0028] Preferably, the reaction temperature in step (2) is 0-55℃ (e.g., 4℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 50℃, 55℃, etc.), the reaction time is 1-10h (e.g., 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 10h, etc.), and the stirring speed is 400-1200rpm (e.g., 400rpm, 600rpm, 800rpm, 1000rpm, 1200rpm, etc.).

[0029] All other point values ​​not listed above within the range of the above values ​​can be selected, and will not be elaborated on here.

[0030] Thirdly, the present invention provides the application of the cationic manganese nanoadjuvant according to the first aspect in the preparation of nanovaccines.

[0031] Fourthly, the present invention provides a nanovaccine, which is obtained by mixing and incubating the cationic manganese nanoadjuvant described in the first aspect with an antigen.

[0032] Preferably, the mass ratio of the cationic manganese nanoadjuvant to the antigen is 1:(0.01-40), for example, 1:0.05, 1:0.1, 1:0.5, 1:1, 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, 1:35, etc.

[0033] Preferably, the antigen includes any one or a combination of at least two of viral proteins, tumor antigens, bacterial antigens, or fungal antigens.

[0034] Preferably, the antigen includes protein antigens and / or polypeptide antigens.

[0035] Preferably, the incubation is carried out in a buffer solution at 20-37℃ (e.g., 20℃, 24℃, 28℃, 30℃, 32℃, 35℃, 37℃, etc.) for 30-90 min (e.g., 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 90 min, etc.).

[0036] Preferably, the buffer solution includes PBS buffer, Tris-HCl buffer, or citrate / citrate buffer.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention achieves precise control over the surface charge and structure of manganese nanoparticles through biomineralization technology, effectively solving key problems in antigen delivery, immune activation, and biocompatibility of traditional vaccine adjuvants. This cationic manganese nanoadjuvant can efficiently adsorb various antigens, forming stable nanovaccine complexes. For naturally negatively charged antigens, its adsorption rate is significantly higher than that of manganese nanoadjuvants. In terms of immune activation, it can significantly upregulate the expression of co-stimulatory molecules on the surface of dendritic cells and promote cytokine secretion, thereby activating the body's innate and adaptive immune responses. Simultaneously, it can effectively target lymph nodes, with higher targeting efficiency than free antigens, increasing antigen accumulation in lymph nodes and further enhancing the immune effect. Furthermore, it exhibits low biotoxicity and lower cytotoxicity than manganese ions. Moreover, it demonstrates excellent stability, with no significant change in particle size after 6 months of storage at 4°C. This adjuvant is suitable for antiviral vaccines, tumor vaccines, and other fields, providing a safe and efficient adjuvant option for the development of novel vaccines. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the preparation process of cationic proteins;

[0040] Figure 2 This is a schematic diagram of the preparation process of cationic manganese nanoadjuvants;

[0041] Figure 3 This is a graph showing the valence state analysis results of manganese in cationic manganese nano-adjuvants;

[0042] Figure 4 This is a graph showing the results of the biotoxicity evaluation of cationic manganese nanoadjuvants on mouse bone marrow-derived dendritic cells;

[0043] Figure 5 This is a graph showing the evaluation results of the activation effect of cationic manganese nanoadjuvants on mouse bone marrow-derived dendritic cells.

[0044] Figure 6 This is a graph showing the in vitro activation effect of cationic manganese nanoadjuvant on mouse bone marrow-derived macrophages (BMDM).

[0045] Figure 7 This is a graph showing the evaluation results of the loading efficiency of OVA by cationic manganese nanoadjuvant;

[0046] Figure 8 This is a graph showing the evaluation results of the loading efficiency of cationic manganese nanoadjuvant on RBD;

[0047] Figure 9 This is a graph showing the evaluation results of the in vitro ability of nanovaccine drugs to promote BMDC antigen presentation.

[0048] Figure 10 This is a graph showing the lymph node targeting evaluation results of nano-vaccine drugs;

[0049] Figure 11 This is a graph showing the evaluation results of the in vivo cellular immune activation effect of nano-vaccine drugs;

[0050] Figure 12 This is a graph showing the in vivo antibody titer determination results for nano-vaccine drugs;

[0051] Figure 13 This is a graph showing the long-term stability evaluation results of cationic manganese nano-adjuvants and nano-vaccine drugs prepared by application examples;

[0052] Figure 14 This is a schematic diagram of the entire preparation process and in vivo immune activation process of the nanovaccine drug of the present invention. Detailed Implementation

[0053] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0054] Example 1

[0055] This embodiment provides a cationic protein, prepared by the following method:

[0056] (1) Add 50 mL of 0.1 mol / L MES buffer to the reaction vessel, add 3 mL of 0.9 mol / L ethylenediamine solution, and stir magnetically to mix.

[0057] (2) Adjust the pH of the mixture to 5.0.

[0058] (3) Add 20 mL of bovine serum albumin (BSA) solution with a concentration of 10 mg / mL dissolved in 0.1 mol / L MES buffer to the above solution and stir continuously at 600 rpm.

[0059] (4) Add 30 mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution with a concentration of 8.90 mg / mL dissolved in 0.1 mol / L MES buffer, and stir at 600 rpm for 2 h at 25 °C.

[0060] (5) The product was collected into a dialysis bag with a molecular weight cutoff of 10 kDa, purified by dialyzing in ultrapure water at 4°C, and then freeze-dried to obtain cationic protein product.

[0061] A schematic diagram of the above preparation process is shown below. Figure 1 As shown.

[0062] The zeta potential of the obtained cationic protein product was characterized as 24.3 ± 1.5 mV, indicating that the protein was successfully cationic modified.

[0063] Example 2

[0064] This embodiment provides a cationic protein, prepared by the following method:

[0065] (1) Add 50 mL of 0.1 mol / L MES buffer to the reaction vessel, add 3 mL of propylenediamine solution, and mix with magnetic stirring.

[0066] (2) Adjust the pH of the mixture to 6.5.

[0067] (3) Add 20 mL of human serum albumin (HSA) solution with a concentration of 50 mg / mL dissolved in 0.1 mol / L MES buffer to the above solution and stir continuously at 1200 rpm.

[0068] (4) Add 30 mL of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) solution dissolved in 0.1 mol / L MES buffer, and stir at 1200 rpm for 12 h at 50 °C.

[0069] (5) The product was collected into a dialysis bag with a molecular weight cutoff of 10 kDa, purified by dialyzing in ultrapure water at 4°C, and then freeze-dried to obtain cationic protein product.

[0070] The zeta potential of the obtained cationic protein product was characterized as 20.6 ± 0.9 mV, indicating that the protein was successfully cationic modified.

[0071] Example 3

[0072] This embodiment provides a cationic protein, prepared by the following method:

[0073] (1) Add 50 mL of 0.1 mol / L MES buffer to the reaction vessel, add 3 mL of 0.9 mol / L pentanediamine solution, and stir magnetically to mix.

[0074] (2) Adjust the pH of the mixture to 4.5.

[0075] (3) Add 20 mL of horse spleen ferritin (AFt) solution with a concentration of 10 mg / mL dissolved in 0.1 mol / L MES buffer to the above solution and stir continuously at 600 rpm.

[0076] (4) Add 30 mL of N,N'-carbonyldiimidazole (CDI) solution dissolved in 0.1 mol / L MES buffer and stir at 600 rpm for 4 h at 25 °C.

[0077] (5) The product was collected into a dialysis bag with a molecular weight cutoff of 10 kDa, purified by dialyzing in ultrapure water at 4°C, and then freeze-dried to obtain cationic protein product.

[0078] The zeta potential of the obtained cationic protein product was characterized as 5.4 ± 1.2 mV, indicating that the protein was successfully cationic modified.

[0079] Example 4

[0080] This embodiment provides a cationic protein, prepared by the following method:

[0081] (1) Add 50 mL of 0.1 mol / L MES buffer to the reaction vessel, add 3 mL of 0.9 mol / L hexamethylenediamine solution, and stir magnetically to mix.

[0082] (2) Adjust the pH of the mixture to 2.5.

[0083] (3) Add 20 mL of mouse serum albumin (OVA) solution with a concentration of 10 mg / mL dissolved in 0.1 mol / L MES buffer to the above solution and stir continuously at 200 rpm.

[0084] (4) Add 30 mL of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) solution dissolved in 0.1 mol / L MES buffer, and stir at 200 rpm for 1 h at 4 °C.

[0085] (5) The product was collected into a dialysis bag with a molecular weight cutoff of 10 kDa, purified by dialyzing in ultrapure water at 4°C, and then freeze-dried to obtain cationic protein product.

[0086] The zeta potential of the obtained cationic protein product was characterized as 12.4 ± 0.6 mV, indicating that the protein was successfully cationic modified.

[0087] Example 5

[0088] This embodiment provides a cationic manganese nanoadjuvant (cMn-1), which is prepared by the following method:

[0089] (1) Add 5 mL of 0.1 mol / L MnCl2·4H2O solution to 10 mL of cationic protein solution prepared in Example 1 with a concentration of 10 mg / mL, and stir at 25 °C for 5 min to obtain a composite solution.

[0090] (2) Adjust the pH to 12 and stir continuously at 600 rpm for 2 hours at 37°C.

[0091] (3) The product was collected into a dialysis bag with a molecular weight cutoff of 100 kDa and purified by dialyzing in ultrapure water overnight. After freeze-drying, the cationic manganese nano-adjuvant product (cMn-1) was obtained.

[0092] A schematic diagram of the above preparation process is shown below. Figure 2 As shown.

[0093] The particle size of the prepared cMn-1 was characterized as 20.6±1.3 nm; the Zeta potential was characterized as 32.9±0.4 mV. The results show that the prepared cationic manganese nanoadjuvant has good dispersibility and uniform size.

[0094] Example 6

[0095] This embodiment provides a cationic manganese nanoadjuvant (cMn-2), which is prepared by the following method:

[0096] (1) Add 5 mL of 1 mol / L manganese nitrate solution to 10 mL of 20 mg / mL cationic protein solution prepared in Example 2, and stir at 25 °C for 5 min to obtain a composite solution.

[0097] (2) Adjust the pH to 12 and stir continuously at 1200 rpm for 12 h at 55 °C.

[0098] (3) The product was collected into a dialysis bag with a molecular weight cutoff of 100 kDa and purified by dialyzing in ultrapure water overnight. After freeze-drying, the cationic manganese nano-adjuvant product (cMn-2) was obtained.

[0099] The particle size of the prepared cMn-2 was characterized as 23.5 ± 0.4 nm; the Zeta potential was characterized as 21.5 ± 0.4 mV. The results show that the prepared cationic manganese nanoadjuvant has good dispersibility and uniform size.

[0100] Example 7

[0101] This embodiment provides a cationic manganese nanoadjuvant (cMn-2), which is prepared by the following method:

[0102] (1) Add 5 mL of 0.1 mol / L manganese sulfate solution to 10 mL of 20 mg / mL cationic protein solution prepared in Example 3, and stir at 25 °C for 5 min to obtain a composite solution.

[0103] (2) Adjust the pH to 10 and stir continuously at 600 rpm for 4 hours at 35°C.

[0104] (3) The product was collected into a dialysis bag with a molecular weight cutoff of 100 kDa and purified by dialyzing in ultrapure water overnight. After freeze-drying, the cationic manganese nano-adjuvant product (cMn-3) was obtained.

[0105] The particle size of the prepared cMn-3 was characterized as 15.7 ± 0.2 nm; the Zeta potential was characterized as 5.3 ± 1.1 mV. The results show that the prepared cationic manganese nanoadjuvant has good dispersibility and uniform size.

[0106] Example 8

[0107] This embodiment provides a cationic manganese nanoadjuvant (cMn-2), which is prepared by the following method:

[0108] (1) Add 5 mL of a mixed solution of 0.1 mol / L manganese chloride and manganese sulfate (molar ratio 1:1) to 10 mL of the cationic protein solution prepared in Example 4 with a concentration of 5 mg / mL, and stir at 25 °C for 5 min to obtain a composite solution.

[0109] (2) Adjust the pH to 10 and stir continuously at 200 rpm for 1 hour at 4°C.

[0110] (3) The product was collected into a dialysis bag with a molecular weight cutoff of 100 kDa and purified by dialyzing in ultrapure water overnight. After freeze-drying, the cationic manganese nano-adjuvant product (cMn-4) was obtained.

[0111] The particle size of the prepared cMn-4 was characterized as 50.2 ± 2.3 nm; the Zeta potential was characterized as 30.2 ± 0.8 mV. The results show that the prepared cationic manganese nanoadjuvant has good dispersibility and uniform size.

[0112] Test Example 1

[0113] Analysis of the valence state of manganese in cationic manganese nano-adjuvants:

[0114] The cMn-1 sample was freeze-dried, ground, compressed into tablets, and then evenly adhered onto adhesive tape. The valence state of Mn was determined using X-ray photoelectron spectroscopy (XPS). Specifically:

[0115] X-ray absorption near-edge structure (XANES) spectra of Mn K-side were acquired at beamline 1W1B of the Beijing Synchrotron Radiation Facility (BSRF) in China. XANES spectra of reference samples (Mn foil, Mn2O3, MnO2, MnO, Mn3O4) and cMn-1 were measured in transmission mode.

[0116] After normalization, the XANES data were processed using IFEFFIT Athena software, and the valence state and existence form of Mn were calculated by least squares fitting.

[0117] See results Figure 3 The cationic manganese nanoadjuvant cMn-1 contains 29% Mn(II) and 71% Mn(III), and its coordination mode is consistent with that of Mn3O4.

[0118] Test Example 2

[0119] Biotoxicity evaluation of cationic manganese nanoadjuvants on mouse bone marrow-derived dendritic cells:

[0120] (1) Culture and extract mouse bone marrow-derived dendritic cells (BMDC).

[0121] (2) BMDC cells were loaded at 5 × 10 4 Cells were seeded in 96-well plates, and different concentrations of cMn-1 nanoadjuvant (0-800 μM) or manganese chloride (MnCl2·4H2O, 0-800 μM) were added, with 3 replicates per group.

[0122] (3) After incubating at 37℃ and 5% CO2 for 24 hours, add 10μL of CCK-8 solution to each well and continue culturing for 2 hours.

[0123] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 450 nm and calculate the cell viability.

[0124] The results are as follows Figure 4 As shown in the figure, the biotoxicity of cMn nano-adjuvant is significantly lower than that of free manganese ions, and it also shows no obvious cytotoxicity to BMDC cells within the dose range of 5 μM.

[0125] Test Example 3

[0126] Evaluation of the activation effect of cationic manganese nanoadjuvant on mouse bone marrow-derived dendritic cells:

[0127] (1) BMDC with 1×10 6 Cells were seeded in 6-well plates and incubated for 24 hours with cMn-1 (2.5 μg / mL), uncationized manganese nanoadjuvant (prepared according to the method in Example 1 of patent CN202110160471.9) (2.5 μg / mL), or PBS (negative control).

[0128] (2) After collecting cells and washing them with flow cytometry buffer, add anti-CD11c-PE, anti-CD80-FITC and anti-CD86-APC antibodies and incubate in the dark for 30 minutes.

[0129] (3) The expression levels of CD80 and CD86 were detected by flow cytometry.

[0130] The results are as follows Figure 5 As shown, even at a concentration of 2.5 μg / mL, cMn nanoadjuvants can still upregulate the expression of CD86 and CD80, efficiently activate BMDCs, and enhance their immune response.

[0131] Test Example 4

[0132] Evaluation of the in vitro activation effect of cationic manganese nanoadjuvant on mouse bone marrow-derived macrophages (BMDM):

[0133] (1) Bone marrow cells from the femur and tibia of C57BL / 6 mice were isolated and induced to differentiate into BMDM in RPMI-1640 medium containing 20 ng / mL M-CSF for 5 days. Then, RPMI-1640 medium containing 20 ng / mL IL-4 was added to induce M2 type BMDM and cultured for 2 days.

[0134] (2) BMDM is 1×10 6 Cells were seeded in 6-well plates, and cMn-1 (5 μg / mL) or PBS (negative control) were added, and incubated for 24 hours.

[0135] (3) Collect cells and stain them with antibodies against CD11b-APC, F4 / 80-PerCP, CD206-FITC, and CD86-PE. Detect surface markers by flow cytometry.

[0136] The results are as follows Figure 6 As shown, cMn nanoadjuvants can promote the expression of CD206 and CD86 significantly higher than those in the PBS group, indicating that they can efficiently activate BMDM and induce pro-inflammatory responses.

[0137] Test Example 5

[0138] Evaluation of antigen loading efficiency of cationic manganese nanoadjuvants:

[0139] (1) The OVA protein was reacted with Cy5 NHS ester in a weakly alkaline buffer and purified by dialysis to obtain Cy5-OVA; the RBD protein was reacted with Cy5 NHS ester in a weakly alkaline buffer and purified by dialysis to obtain Cy5-RBD.

[0140] (2) cMn-1 nano-adjuvant or uncationized manganese nano-adjuvant (prepared according to the method of Example 1 in patent CN202110160471.9) was mixed with Cy5-OVA at different mass ratios and incubated at 25°C for 1 hour; cMn-1 nano-adjuvant or uncationized manganese nano-adjuvant (abbreviated as Mn) was mixed with Cy5-RBD at a mass ratio (1:10) and incubated at 37°C for 2 hours.

[0141] (3) Centrifuge at 30,000 rpm for 30 minutes, take the supernatant and measure the fluorescence intensity of the unbound antigen using a fluorescence spectrophotometer, and prepare a standard curve for fluorescence measurement using gradient concentrations of Cy5-OVA or Cy5-RBD.

[0142] (4) Calculate the adsorption rate. The formula for calculating the adsorption rate is: Adsorption rate (%) = (1 - fluorescence intensity of supernatant / initial fluorescence intensity) × 100.

[0143] The results are as follows Figure 7 and Figure 8 As shown, the adsorption rates of cationic manganese nanoadjuvants for OVA and RBD are significantly higher than those of uncationized manganese nanoadjuvants, indicating that cationic manganese nanoadjuvants can effectively adsorb and carry antigen molecules, improve antigen loading efficiency, and obtain nanovaccine drugs constructed by combining cationic manganese nanoadjuvants with antigens.

[0144] Application examples

[0145] This application example provides a nano-vaccine drug (cMnVax), which is prepared by the following method:

[0146] The cMn-1 nano-adjuvant prepared in Example 5 was mixed with OVA antigen at a mass ratio of 25:10 and incubated in PBS buffer at 25°C for 60 min. The antigen was the model antigen OVA, and the mass ratio was the ratio of the mass of manganese in the cMn-1 nano-adjuvant to the mass of the antigen.

[0147] A schematic diagram of the entire preparation process and in vivo immune activation process of the nano-vaccine drug of the present invention is shown below. Figure 14 As shown.

[0148] Test Example 6

[0149] Evaluation of the in vitro ability of nanovaccine drugs to promote BMDC antigen presentation:

[0150] (1) BMDC with 1×10 6 Cells were seeded in 6-well plates, and nanovaccine drugs (manganese concentration of 2.5 μg / mL) or PBS were added and incubated for 24 hours.

[0151] (2) Add CD11c, MHC-I, and MHC-II antibodies for staining.

[0152] (3) Flow cytometry was used to detect surface markers.

[0153] The results are as follows Figure 9 As shown, the MHC-I and MHC-II surface markers of BMDC in the nanovaccine drug group were significantly increased, indicating that the nanovaccine drug can enhance the antigen presentation ability of BMDC.

[0154] Test Example 7

[0155] Evaluation of lymph node targeting of cationic manganese nanoadjuvants:

[0156] (1) Select 6-8 week old BALB / c mice and subcutaneously inject Cy5-labeled cMn or PBS (negative control, Ctr).

[0157] (2) Dissect the mice to obtain lymph nodes, and use IVIS Spectrum in vivo imaging system to detect lymph node fluorescence signals 24 hours after injection.

[0158] The results are as follows Figure 10 As shown, the fluorescence intensity of the nano-adjuvant group in the lymph node site was higher than that of the Ctr group, indicating that the nano-adjuvant significantly improved the delivery efficiency to the lymph node.

[0159] Test Example 8

[0160] Evaluation of the in vivo cellular immune activation effect of nano-vaccine drugs:

[0161] (1) Select BALB / c mice aged 6-8 weeks and subcutaneously inject Cy5-labeled cMn-OVA (10μg OVA / 25μg cMn, preparation method as described in test example 5) or free Cy5-OVA (negative control, Ctr).

[0162] (2) Seven days after immunization, mice were sacrificed and spleen single-cell suspensions were prepared and stimulated with OVA protein (5 μg / mL) for 24 hours.

[0163] (3) Flow cytometry detection of CD4 + and CD8 + The level of intracellular IFN-γ in T cells.

[0164] The results are as follows Figure 11 As shown, the nano-vaccine drug group CD4 + IFN-γ + and CD8 + IFN-γ + The proportion of cells was significantly higher than that in the free OVA group, indicating that the nanovaccine drug effectively activated the cellular immune response.

[0165] Test Example 9

[0166] In vivo antibody titer determination of nano-vaccine drugs:

[0167] (1) Animal immunization: BALB / c mice were randomly divided into two groups of 4 mice each. Primary immunization: mice in the nanovaccine group were subcutaneously injected with 100 μL of nanovaccine (containing 10 μg of OVA antigen); mice in the free OVA group were subcutaneously injected with 100 μL of 10 μg of OVA.

[0168] (2) Boost immunization: immunize again on day 14 after the initial immunization.

[0169] (3) Serum collection: On day 21 after the initial immunization, blood was collected from mice by orbital blood collection method, left to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm for 15 minutes to separate the serum, which was then stored at -20℃ for later use.

[0170] (4) ELISA method to determine antibody titer.

[0171] The results are as follows Figure 12 As shown, nano-vaccine drugs can effectively enhance the immunogenicity of OVA antigens and stimulate the body to produce a highly efficient humoral immune response.

[0172] Test Case 10

[0173] Long-term stability evaluation of cationic manganese nano-adjuvants and nano-vaccine drugs:

[0174] (1) Store the cationic manganese nanoadjuvant and nanovaccine drug at 4°C and take samples every 30 days.

[0175] (2) DLS determination of hydrated particle size.

[0176] The results are as follows Figure 13 As shown in the figure, the cationic manganese nano-adjuvant and nano-vaccine drug involved in this invention showed no significant change in particle size within 6 months of storage at 4°C, demonstrating good long-term stability.

[0177] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0178] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0179] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A cationic manganese nanoadjuvant, characterized in that, The cationic manganese nanoadjuvant comprises manganese tetroxide nanoparticles and cationic template molecules coated on the surface of the manganese tetroxide nanoparticles. The cationic template molecules are proteins, protein fragments thereof, or polypeptide fragments thereof that have undergone cationic modification.

2. The cationic manganese nanoadjuvant according to claim 1, characterized in that, The molar ratio of the cationic template molecule to manganese is 1:(10-1000).

3. The cationic manganese nanoadjuvant according to claim 1 or 2, characterized in that, The protein includes any one or a combination of at least two of the following: bovine serum albumin, human serum albumin, mouse serum albumin, transferrin, horse spleen ferritin, human recombinant heavy chain ferritin, or antigenic protein.

4. The cationic manganese nanoadjuvant according to any one of claims 1-3, characterized in that, The cationization modification process involves coupling the cationizing agent to the template molecule using a coupling agent. Preferably, the cationizing modifier includes any one or a combination of at least two of ethylenediamine, propylenediamine, butylenediamine, pentanediamine, or hexamethylenediamine; Preferably, the coupling agent comprises any one or a combination of at least two of the following: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-carbonyldiimidazole, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, and benzotriazine-1-oxytripyrrolidinylphosphonium hexafluorophosphate; Preferably, the molar ratio of the template molecule to the coupling agent is 1:(1-3000); Preferably, the molar ratio of the template molecule to the cationic modifier is 1:(1-3000).

5. The method for preparing cationic manganese nanoadjuvant according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Dissolve the template molecule in a buffer solution, mix it with a coupling agent and a cationizing agent, react it under stirring, and purify the product by dialysis or ultrafiltration to obtain the cationized template molecule. (2) Mix the aqueous solution of divalent manganese salt with the cationic template molecule solution to obtain a composite solution; adjust the pH of the composite solution to alkaline, react under stirring, and purify the product by dialysis or ultrafiltration to obtain the cationic manganese nanoadjuvant.

6. The preparation method according to claim 5, characterized in that, The concentration of the template molecule dissolved in the buffer solution in step (1) is 1-50 mg / mL; Preferably, the reaction in step (1) is carried out at pH 2-7, at a temperature of 0-50℃, for a time of 0.5-12h, and at a stirring speed of 200-1200rpm.

7. The preparation method according to claim 5 or 6, characterized in that, The divalent manganese salt in step (2) includes any one or a combination of at least two of manganese chloride, manganese nitrate, manganese acetate, or manganese sulfate; Preferably, the concentration of the divalent manganese salt aqueous solution is 0.1-1 mol / L; Preferably, the concentration of the cationized template molecule solution is 1-20 mg / mL; Preferably, the pH of the composite solution is adjusted to 9-12; Preferably, the pH of the composite solution is adjusted by an alkaline reagent, which includes any one or a combination of at least two of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, triethylamine, pyridine, N-methylmorpholine, or tetramethylethylenediamine. Preferably, the reaction temperature in step (2) is 0-55℃, the reaction time is 1-10h, and the stirring speed is 400-1200rpm.

8. The application of the cationic manganese nanoadjuvant according to any one of claims 1-4 in the preparation of nanovaccines.

9. A nano-vaccine, characterized in that, The nanovaccine is obtained by mixing and incubating the cationic manganese nanoadjuvant according to any one of claims 1-4 with the antigen.

10. The nanovaccine according to claim 9, characterized in that, The mass ratio of the cationic manganese nanoadjuvant to the antigen is 1:(0.01-40); Preferably, the antigen includes any one or a combination of at least two of viral proteins, tumor antigens, bacterial antigens, or fungal antigens; Preferably, the antigen includes protein antigens and / or polypeptide antigens; Preferably, the incubation is carried out in a buffer solution at 20-37°C for 30-90 minutes; Preferably, the buffer solution includes PBS buffer, Tris-HCl buffer, or citrate / citrate buffer.

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