Construction of a protein-metal ion-based antigenic peptide nanovaccine and its application as a tumor vaccine

Abstract

This invention provides a protein-metal ion-based antigenic peptide nanovaccine, wherein the nanovaccine is formed by loading antigenic peptides onto protein-metal ion composite nanoparticles; the metal ion is magnesium ion or zinc ion, which are derived from magnesium chloride or zinc chloride, respectively; the protein includes at least one of human hemoglobin, human serum albumin, and bovine serum albumin; and the antigen includes tumor-associated antigens and neoantigens.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and bioengineering, specifically to the construction of a protein-metal ion-based antigen peptide nanovaccine and its application as a tumor vaccine. Background Technology

[0002] In clinical oncology treatment, tumor immunotherapy, with its advantages of high specificity and few side effects, is one of the most promising approaches to cancer treatment. Currently, various tumor immunotherapy methods exist, including immune checkpoint inhibitor therapy, adoptive cell immunotherapy, and cancer vaccines. With the development of next-generation gene sequencing and bioinformatics, individualized identification of tumor neoantigens has become possible, and personalized neoantigen vaccines have begun to show promising efficacy in small-scale early-stage clinical trials. Nevertheless, personalized neoantigen immunotherapy for tumors still faces significant challenges, such as poor neoantigen lymphocyte homing ability, low presentation efficiency, and weak antigen immunogenicity.

[0003] In recent decades, nanotechnology has gradually become a focus of attention in the field of anti-tumor immunotherapy. Using nanocarriers to load immunotherapeutic drugs can improve drug solubility and increase their circulation time in vivo. Simultaneously, the structural characteristics of nanocarriers and lymphatic organs make it easier for nanoparticles to concentrate in immune organs such as lymph nodes and the spleen. Loading neoantigens onto nanocarriers allows for more precise delivery of neoantigens to immune cells such as dendritic cells (DCs) and T lymphocytes (T cells).

[0004] Cancer vaccines, which utilize antigen-presenting cells (APCs) to recognize tumor antigens and stimulate T cells in the body, thereby inducing a sustained and robust immune response to prevent and treat cancer, represent a novel strategy with significant immunotherapeutic potential. The active ingredients of cancer vaccines consist of four components: a delivery carrier, a tumor antigen, an immune adjuvant, and the formulation. Therefore, effectively enhancing the immunogenicity of the antigen and the activation effect of the adjuvant are two main strategies in vaccine development.

[0005] Inorganic adjuvants such as aluminum, monophospholipid A (MPL), and squalene oil-in-water emulsion (MF59) are currently the most widely used vaccine adjuvants for enhancing the immunogenicity of vaccine candidates. However, after the free antigen and adjuvant enter the human body, they target different APCs, resulting in the adjuvant's ineffectiveness against cancer cells and the inability to effectively amplify the immunogenicity of intracellular antigens. Simultaneously, the size limitation of lymph nodes prevents the precise delivery of vaccines to immune cells or lymphoid tissues.

[0006] Therefore, simultaneously loading antigens and adjuvants into functional nanomaterials offers unique advantages. To date, most nanomaterials have carried antigens through covalent bonding, electrostatic interactions, or cross-linking agents to promote antigen uptake by immune cells. However, the antigen-carrying capacity of nanomaterials is not ideal, hindering practical applications.

[0007] In conclusion, there is an urgent need for a universal, lymph node-targeting tumor vaccine that promotes efficient antigen delivery in lymph nodes and activates adaptive and innate immune responses. Summary of the Invention

[0008] This invention addresses the shortcomings of existing technologies by providing an antigenic peptide nanovaccine based on protein-metal ions.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A protein-metal ion-based antigenic peptide nanovaccine, wherein the nanovaccine is formed by loading antigenic peptides onto protein-metal ion composite nanoparticles;

[0011] The metal ion is a magnesium ion or a zinc ion, which is derived from magnesium chloride or zinc chloride, respectively.

[0012] The protein includes at least one of human hemoglobin, human serum albumin, and bovine serum albumin;

[0013] The antigens include tumor-associated antigens and neoantigens.

[0014] To optimize the above technical solution, the specific measures also include:

[0015] Furthermore, the molar ratio of protein to metal ions is 1:650.

[0016] Furthermore, the antigenic peptide is connected to the protein-metal ion composite nanoparticles through electrostatic interactions or chemical bonds.

[0017] Furthermore, the protein-metal ion composite nanoparticles have a water content and a particle size of 100–200 nm.

[0018] A method for preparing an antigenic peptide nanovaccine based on the above-mentioned protein-metal ion includes the following steps:

[0019] S1. Dissolve protein molecules and, under alkaline conditions with a pH of 10-12, chelate protein molecules with metal ions through biomineralization to form protein-metal ion composite nanoparticles (HemoM / HemoZ nanoparticles).

[0020] S2. Dissolve the protein-metal ion composite nanoparticles and antigenic peptides obtained in step S1 in water or PBS buffer, and incubate them to obtain nanovaccines (HemoMap / HemoZap nanovaccines, HSAMap / HSAZap nanovaccines).

[0021] Furthermore, in step S1, the concentration of metal ions is 0.1 mol / L, the concentration of protein molecules is 10 mg / mL, and the selected protein surface carries a negative charge, which can better control the stability of the protein structure and is beneficial to maintaining the stability of the protein-metal ion composite nanoparticles formed subsequently.

[0022] Further, in step S1, the reaction is a stirring reaction, the stirring temperature is 40-50℃, the stirring time is 3-5 hours, and the stirring rate is 100-1000 r / min.

[0023] Furthermore, in step S2, the mass ratio of the antigen peptide to the protein-metal ion composite nanoparticles is 1:0.2 to 1.

[0024] Furthermore, in step S2, the incubation temperature of the protein-metal ion composite nanoparticles and the antigen peptide is 25-37℃, and the incubation time is 1-3 hours.

[0025] The aforementioned protein-metal ion-based antigenic peptide nanovaccines are used to prepare tumor vaccines.

[0026] The beneficial effects of this invention are:

[0027] Human hemoglobin and human serum albumin are proteins composed of 574 and 585 amino acids, respectively, with molecular weights of approximately 64.5 kDa and 66.5 kDa. They are natural proteins derived from the human body, exhibiting high biocompatibility, no immunogenicity, and abundant free carboxyl and amino groups, making them easily modifiable. This invention provides a method for constructing a protein-metal ion-based antigenic peptide nanovaccine: by dissolving human hemoglobin or human serum albumin molecules, the protein molecules are chelated with metal ions through biomineralization under alkaline conditions, forming protein-metal ion composite nanoparticles; the protein-metal ion composite nanoparticles and antigenic peptides obtained in the above steps are then dissolved in water or PBS buffer, and incubated to obtain the nanovaccine. This synthetic method is simple, uses inexpensive and readily available raw materials, and does not involve complex chemical reactions or difficult-to-remove intermediate reaction products.

[0028] The nanovaccines prepared using the above methods can efficiently accumulate in tumor-draining lymph nodes, promoting antigen presentation, enhancing the uptake efficiency of antigens by immune cells, and strengthening anti-tumor effects. They induce a highly efficient cellular immune response with relatively low antigen doses and injection frequency. Secondly, the protein-metal ion composite nanoparticles in the constructed nanovaccines can stably load antigen molecules. Finally, the components of the HemoMap / HemoZap nanovaccine based on HemoM / HemoZ nanoparticles exhibit good biocompatibility, and fluorescence imaging indicates that they have virtually no toxic side effects, greatly increasing the possibility of further clinical research. Attached Figure Description

[0029] Figure 1 Flowchart of HemoMap / HemoZap nanovaccine preparation;

[0030] Figure 2 Transmission electron microscope images and elemental distribution images of HemoM nanoparticles;

[0031] Figure 3 The graph shows the cytotoxicity evaluation of HemoM nanoparticles and the statistical results of the in vitro experiment promoting the maturation of BMDCs.

[0032] Figure 4 Hydration particle size and transmission electron microscope images of HemoM nanoparticles and HemoMap nanovaccines.

[0033] Figure 5 The images show the lymph node targeting fluorescence imaging, quantitative fluorescence analysis of inguinal and axillary lymph nodes, and flow cytometry data of antigen internalization for the HemoMap nanovaccine.

[0034] Figure 6 Images of HemoMap nanovaccine in vivo anti-tumor experiments, tumor inhibition effect evaluation, and mouse survival time.

[0035] Figure 7 Flowchart of HSAMap / HSAZap nanovaccine preparation;

[0036] Figure 8 Transmission electron microscope images and elemental distribution images of HSAM nanoparticles;

[0037] Figure 9 Statistical graphs showing the results of cytotoxicity evaluation of HSAM nanoparticles and in vitro experiments promoting BMDC maturation;

[0038] Figure 10 The images show the lymph node-targeted fluorescence imaging and fluorescence distribution in major organs of the HSAMap nanovaccine.

[0039] Figure 11The images show in vivo anti-tumor experiments of the HSAMap nanovaccine, evaluation of tumor inhibition effect, and mouse survival time.

[0040] Figure 12 The graph shows the cytotoxicity evaluation of HemoZ nanoparticles and the statistical results of in vitro activation of BMDCs and T cells. Detailed Implementation

[0041] Examples 1-5 use HemoM nanoparticles formed from magnesium ions and HemoMap nanovaccines formed with Tyrp1, a highly expressed antigenic peptide in mouse melanoma, as examples.

[0042] Examples 6-10 use HSAM nanoparticles formed by magnesium ions and the HSAMap nanovaccine formed by them with the mouse melanoma high-expression antigenic peptide M27 as examples;

[0043] Examples 11-12 illustrate that zinc ion-formed nanoparticles also exhibit immune cell stimulation at safe dosages. The preparation and application of other protein molecules (such as bovine serum albumin), other magnesium / zinc ion-formed nanoparticles, and nanovaccines formed from other tumor antigens are essentially the same and will not be described in detail elsewhere.

[0044] Example 1: Preparation and Characterization of HemoM Nanoparticles

[0045] HemoM nanoparticles were prepared by the following methods, such as Figure 1 As shown, the specific steps are as follows:

[0046] S1. Prepare a 10 mg / mL human hemoglobin solution (HB) and stir at 50 °C for 2 h;

[0047] S2. Add 500 μL of 1.0 M MgCl2 to HB solution, stir at 50 °C for 20 min, then add 1 mL of 1.0 M NaOH to adjust the pH to 12, continue stirring for 3 h, and obtain HemoM nanoparticles by dialysis purification and freeze-drying.

[0048] HemoM nanoparticles were digested with nitric acid, and the concentrations of magnesium and iron in the HemoM nanoparticles were detected by inductively coupled plasma optical emission spectrometry (ICP-OES). The results showed that 1 mg of HemoM nanoparticles contained 1.7% magnesium and 0.07% iron.

[0049] like Figure 2 As shown, the HemoM nanoparticles have an average particle size of 31.25 nm, are well dispersed, and have uniform size; the elemental distribution image shows that magnesium in the HemoM nanoparticles is concentrated in the core of the nanoparticles.

[0050] Example 2: Cytotoxicity evaluation of HemoM nanoparticles and assay for activation of BMDC cells

[0051] Cytotoxicity evaluation: Bone marrow-derived BMDCs were seeded in 96-well plates at a density of 5000 cells / well on day 6 and cultured for 24 h. Then, they were incubated with MgCl2, HB solution or HemoM nanoparticles for 24 h, respectively. Cell viability was assessed using the CCK-8 assay kit.

[0052] Flow cytometry analysis of activated BMDCs cell viability assay: First, C57BL / 6 mice were euthanized by cervical dislocation, and bone marrow was aseptically extracted. Red blood cells were lysed to prepare a single-cell suspension. Then, the cells were cultured in complete medium (containing 10% fetal bovine serum and penicillin-streptomycin) at 20 ng / mL containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng / mL interleukin-4 (IL-4). The medium was changed 3 / 4 on days 2 and 4. On day 6, bone marrow-derived BMDCs were sputtered at a rate of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of cells / well in 96-well plates and cultured for 24 h. Then, 100 μL of each well was added to each well of the following solutions: *Panax notoginseng* physiological saline, *Panax notoginseng* LPS (1 μg / mL), HB solution (3.0 mg / mL), MgCl2·6H2O (2.0 mM), or HemoM nanoparticles. After incubation for 48 h, cells were collected on day 10 and stained with CD11c-FITC antibody, CD80-APC antibody, and CD86-PE antibody (all purchased from Biolegend) for flow cytometry analysis (CytoFLEX, Beckman, USA).

[0053] like Figure 3 As shown, compared with free magnesium ions, HemoM nanoparticles did not exhibit significant cytotoxicity to BMDCs even at a concentration of 2.0 mM; at the same time, HemoM nanoparticles enhanced the stimulatory effect of neoantigen peptides on BMDCs, and HemoM induced higher levels of BMDC maturation.

[0054] Example 3: Construction of HemoMap nanovaccine using HemoM nanoparticles combined with the neoantigen Tyrp1

[0055] The specific steps for constructing the HemoMap nanovaccine by combining the HemoM nanoparticles obtained in Example 1 with the neoantigen Tyrp1 are as follows:

[0056] S1. The mouse melanoma-expressing antigenic peptide Tyrp1 (amino acid sequence: CTAPDNLGYM) was selected as the immunogenic research object; 3.0 mg / mL HemoM nanoparticles and Tyrp1 were incubated at 25℃ for 3 h to obtain a mixed solution; the mass ratio of magnesium in the antigenic peptide Tyrp1 to HemoM nanoparticles was 1:0.25.

[0057] S2. Centrifuge the mixed solution obtained in step S1 at 12000 rpm for 20 min, and measure the concentration of unbound antigen in the supernatant by HPLC.

[0058] like Figure 4 As shown, the hydrated particle size of the HemoMap nanovaccine formed after binding with Tyrp1 increased significantly, indicating that the Tyrp1 antigen and HemoM nanoparticles were effectively bound together.

[0059] Example 4: HemoMap Nanoparticle Vaccine Fluorescence Imaging

[0060] S1. Under the premise of complying with national animal protection agreements, 4-6 week old C57BL / 6 mice were selected and divided into 2 groups of 3 mice each. The following reagents were injected subcutaneously into the groin: a) Tyrp1 antigen modified with 20 μg of fluorescent molecule Cy5-NHS; b) HemoMap nanovaccine constructed from 300 μg of HemoM nanoparticles carrying Tyrp1 antigen modified with 20 μg of fluorescent molecule Cy5-NHS.

[0061] S2. With the injection time point as 0h, after fluorescence imaging at 24, 48, 72, and 96h, the axillary and inguinal lymph nodes of mice were removed for fluorescence imaging analysis, and the fluorescence of the inguinal draining lymph nodes was quantitatively analyzed.

[0062] S3. Flow cytometry was used to analyze dendritic cells (DCs) in lymph node tissue 48 hours after injection, and the internalization of Tyrp1 antigen in DCs was statistically analyzed.

[0063] like Figure 5 As shown, compared with the Tyrp1 antigen alone, the HemoMap nanovaccine induced a stronger fluorescence signal in lymph nodes; quantitative analysis showed that 48 hours after injection, the accumulation efficiency of the nanovaccine in lymph nodes was 2.5 times that of the free Tyrp1 antigen, indicating that the HemoMap nanovaccine promotes the delivery and effective accumulation of antigen to lymph nodes in vivo.

[0064] Example 5: Evaluation of the therapeutic effect of HemoMap nanovaccine on melanoma

[0065] S1. Under the premise of complying with national animal protection agreements, 4-6 week old C57BL / 6 mice were selected and divided into 4 groups of 6 mice each. The following reagents were injected subcutaneously into the groin: a) 20 μg Tyrp1 antigen without HemoM nanoparticles; b) HemoM nanoparticles; c) 300 μg HemoMap nanovaccine carrying 20 μg Tyrp1 antigen; d) physiological saline.

[0066] S2, Mouse tumor model: B16F10 melanoma cells resuspended at 1×10⁻⁶ 6 After administering 100 μL per mL, each mouse was subcutaneously inoculated with 10 [units / mL]. 5 One cell, treatment to begin in three days;

[0067] S3, Mouse Immunization: Mice were vaccinated three times, with a subcutaneous injection of 1×10⁻⁶ mg / L. 5 The first vaccination of B16F10 cells was on day 0, the second vaccination was on day 3, the third vaccination was on day 5, and the third vaccination was on day 7.

[0068] S4. Record tumor size 3-4 times per week using calipers. Tumor volume is calculated as length × width × width / 2. When the mouse tumor volume is greater than 1500 mm², the tumor size is recorded. 3 The mice were euthanized, and their lifespan was recorded.

[0069] like Figure 6 As shown, the HemoMap nanovaccine can inhibit the growth of melanoma in mice and prolong their survival.

[0070] Example 6: Preparation and Characterization of HSAM Nanoparticles

[0071] HSAM nanoparticles are prepared by the following methods, such as Figure 7 As shown, the specific steps are as follows:

[0072] S1. Prepare a 10 mg / mL human serum albumin solution (HSA) and stir at 50 °C for 2 h;

[0073] S2. Add 500 μL of 1.0 M MgCl2 to HSA solution, stir at 50 °C for 20 min, then add 1 mL of 1.0 M NaOH to adjust the pH to 10, continue stirring for 3 h, and obtain HSAM nanoparticles after dialysis purification and freeze-drying.

[0074] HSAM nanoparticles were digested with nitric acid, and the magnesium concentration in the HSAM nanoparticles was detected by inductively coupled plasma optical emission spectrometry (ICP-OES). The results showed that 1 mg of HSAM nanoparticles contained 1.4% magnesium.

[0075] like Figure 8As shown, the average particle size of the HSAM nanoparticles is 45.8 nm, and they are well dispersed and uniform in size; the elemental distribution image shows that the magnesium element in the HSAM nanoparticles is uniformly distributed in the albumin nanospheres.

[0076] Example 7: Construction of HSAMap Nanovaccine by Combining HSAM Nanoparticles with Neoantigen M27

[0077] The specific steps for constructing the HemoMap nanovaccine by combining the HSAM nanoparticles obtained in Example 6 with the neoantigen M27 are as follows:

[0078] S1. Mouse melanoma-expressing antigenic peptide M27 (amino acid sequence: LCPGNKYEM) was selected as the immunogenic research object. 2 mg of antigenic peptide M27 was added to 2 mL of PBS solution. EDC and NHS were added at a molar ratio of antigenic peptide:EDC:NHS of 1:2:2, and the mixture was incubated for 15 min. Then, 10 mg of HSAM nanoparticles were added, and the reaction was allowed to proceed for 2 h. The final reaction was dialyzed for 5 h, lyophilized, and stored at -80℃.

[0079] S2. Centrifuge the mixed solution obtained in step S1 at 12000 rpm for 20 min, and measure the concentration of unbound antigen in the supernatant by HPLC.

[0080] The hydrated particle size of the HSAMap nanovaccine formed after binding with M27 increased to 58.44 nm, indicating that the M27 antigen and HemoM nanoparticles were effectively bound together.

[0081] Example 8: Cytotoxicity evaluation of HSAMap nanovaccine and assay for activation of BMDC cells.

[0082] Cytotoxicity evaluation: Bone marrow-derived BMDCs were seeded in 96-well plates at a density of 5000 cells / well on day 6 and cultured for 24 h. Then, they were incubated with different concentrations of HSAMap nanovaccine for 24 h, and cell viability was assessed using the CCK-8 assay kit.

[0083] Flow cytometry analysis of activated BMDCs cell viability assay: First, C57BL / 6 mice were euthanized by cervical dislocation, and bone marrow was aseptically extracted. Red blood cells were lysed to prepare a single-cell suspension. Then, the cells were cultured in complete medium (containing 10% fetal bovine serum and penicillin-streptomycin) at 20 ng / mL containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng / mL interleukin-4 (IL-4). The medium was changed 3 / 4 on days 2 and 4. On day 6, bone marrow-derived BMDCs were sputtered at a rate of 1×10⁻⁶ cells / mL. 5Cells were seeded at a density of cells / well in 96-well plates and cultured for 24 h. Then, 100 μL of each well of the cells were treated with yin-type saline, yang-type LPS (1 μg / mL), HSAM nanoparticles, and HSAMap nanovaccine and incubated for 48 h. On day 10, the cells were collected and stained with CD11c-FITC antibody, CD80-APC antibody, and CD86-PE antibody (all purchased from Biolegend) for flow cytometry analysis (CytoFLEX, Beckman, USA).

[0084] like Figure 9 As shown, the HSAMap nanovaccine had no significant cytotoxicity to BMDCs; HSAM nanoparticles enhanced the stimulatory effect of neoantigen peptides on BMDCs, and HSAM nanoparticles induced higher levels of BMDC maturation.

[0085] Example 9: HSAMap Nanoparticle Vaccine Fluorescence Imaging

[0086] S1. Under the premise of complying with national animal protection agreements, 4-6 week old C57BL / 6 mice were selected and divided into 2 groups of 3 mice each. The following reagents were injected subcutaneously into the groin: a) M27 antigen modified with 20 μg of fluorescent molecule Cy5; b) HSAMap nanovaccine constructed from 200 μg of HSAM nanoparticles modified with 20 μg of fluorescent molecule Cy5.

[0087] S2. With the injection time point as 0h, after fluorescence imaging at 2, 4, 8, 24, 48, 72, and 96h, the axillary and inguinal lymph nodes of mice were removed for fluorescence imaging analysis, and the fluorescence of the inguinal draining lymph nodes was quantitatively analyzed.

[0088] like Figure 10 As shown, compared with the M27 antigen alone, the HSAMap nanovaccine induced a stronger fluorescence signal in lymph nodes; quantitative analysis showed that 96 hours after injection, the accumulation efficiency of the nanovaccine in lymph nodes was 1.5 times that of the free M27 antigen, indicating that the HSAMap nanovaccine promotes the delivery and effective accumulation of antigen to lymph nodes in vivo.

[0089] Example 10: Evaluation of the therapeutic effect of HSAMap nanovaccine on melanoma

[0090] S1. Under the premise of complying with national animal protection agreements, 4-6 week old C57BL / 6 mice were selected and divided into 4 groups of 6 mice each. The following reagents were injected subcutaneously into the groin: a) 20 μg of M27 antigen without HSAM nanoparticles; b) HSAM nanoparticles; c) 300 μg of HSAMap nanovaccine carrying 20 μg of M27 antigen; d) physiological saline.

[0091] S2, Mouse tumor model: B16F10 melanoma cells resuspended at 1×10⁻⁶ 6 After administering 100 μL per mL, each mouse was subcutaneously inoculated with 10 [units / mL]. 5 One cell, treatment to begin in three days;

[0092] S3, Mouse Immunization: Mice were vaccinated three times, with a subcutaneous injection of 1×10⁻⁶ mg / L. 5 The first vaccination of B16F10 cells was on day 0, the second vaccination was on day 3, the third vaccination was on day 5, and the third vaccination was on day 7.

[0093] S4. Record tumor size 3-4 times per week using calipers. Tumor volume is calculated as length × width × width / 2. When the mouse tumor volume is greater than 1500 mm², the tumor size is recorded. 3 The mice were euthanized, and their lifespan was recorded.

[0094] like Figure 11 As shown, the HSAMap nanovaccine can inhibit the growth of melanoma in mice and prolong their survival.

[0095] Example 11 Preparation of HemoZ Nanoparticles

[0096] The specific preparation method of HemoZ nanoparticles is as follows:

[0097] S1. Prepare a 10 mg / mL human hemoglobin solution (HB) and stir at 50 °C for 2 h;

[0098] S2. Add 500 μL of 1.0 M ZnCl2 to HB solution, stir at 50 °C for 20 min, then add 1 mL of 1.0 M NaOH to adjust the pH to 12, continue stirring for 3 h, and obtain HemoZ nanoparticles after dialysis purification and freeze-drying.

[0099] Example 12: Activation effect of HemoZ nanoparticles on DC and T cells

[0100] Cytotoxicity evaluation: Human umbilical vein endothelial cells were used as normal tissue cells and seeded in 96-well plates at a density of 5000 cells / well. After culturing for 24 h, they were incubated with different concentrations of HemoZ nanoparticles for 24 h. Cell viability was assessed using the CCK-8 assay kit.

[0101] Flow cytometry analysis of activated BMDCs cell viability assay: First, C57BL / 6 mice were euthanized by cervical dislocation, and bone marrow was aseptically extracted. Red blood cells were lysed to prepare a single-cell suspension. Then, the cells were cultured in complete medium (containing 10% fetal bovine serum and penicillin-streptomycin) at 20 ng / mL containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng / mL interleukin-4 (IL-4). The medium was changed 3 / 4 on days 2 and 4. On day 6, bone marrow-derived BMDCs were sputtered at a rate of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of cells / well in 96-well plates and cultured for 24 h. Then, 100 μL of each well was added to each well of the *Panax notoginseng* saline, *Panax notoginseng* LPS (1 μg / mL), and HemoZ nanoparticles (maximum safe dose) for incubation. After incubation for 48 h, cells were collected on day 10 and stained with CD11c-FITC antibody, CD80-APC antibody, and CD86-PE antibody (all purchased from Biolegend) for flow cytometry analysis (CytoFLEX, Beckman, USA).

[0102] Flow cytometry analysis of activated spleen T cell activity assay: First, C57BL / 6 mice were euthanized by cervical dislocation, and the spleen was aseptically removed. After grinding and lysing red blood cells, a single-cell suspension was prepared. The cells were then cultured in complete medium (90% 1640 + 10% FBS + 1% P / S) with added saline and HemoZ nanoparticles (maximum safe dose). After incubation for 48 h, cells were collected and stained with CD3-FITC antibody, CD8-PerCp / Cy5 antibody, CD25-APC antibody, and CD69-PE antibody (all purchased from Biolegend) for flow cytometry analysis (CytoFLEX, Beckman, USA).

[0103] like Figure 12 As shown, HemoZ nanoparticles are at a safe dose of 100 μmol / L; at this safe dose, HemoZ increased the proportion of mature DCs by 3 times; and increased the proportions of early (CD69) and late (CD25) activation markers of T cells by 3.4 times and 2.5 times, respectively.

[0104] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A protein-metal ion-based antigenic peptide nanovaccine, characterized by, The nano-vaccine is a nano-vaccine formed by loading antigen peptides on protein-metal ion complex nanoparticles; The metal ion is a magnesium ion or a zinc ion, and the magnesium ion or the zinc ion is derived from magnesium chloride or zinc chloride, respectively. The protein includes at least one of human hemoglobin, human serum albumin and bovine serum albumin. The antigen includes a tumor-associated antigen and a neoantigen. The protein molecules are chelated with the metal ions by biomineralization under alkaline conditions with a pH value of 10-12, and a protein-metal ion complex nanoparticle is formed by the reaction; and the antigen peptides are connected to the protein-metal ion complex nanoparticle by electrostatic interaction or chemical bonds.

2. The protein-metal ion-based antigenic peptide nanovaccine according to claim 1, characterized in that, The molar ratio of the protein to the metal ion is 1:

650.

3. The protein-metal ion-based antigenic peptide nanovaccine according to claim 1, characterized in that, The hydration particle size of the protein-metal ion complex nanoparticle is 100-200 nm.

4. A method for preparing the protein-metal ion-based antigenic peptide nanovaccine according to any one of claims 1 to 3, characterized by, The method comprises the following steps: S1, dissolving protein molecules, chelating the protein molecules with the metal ions by biomineralization under alkaline conditions with a pH value of 10-12, and forming a protein-metal ion complex nanoparticle by the reaction; S2, dissolving the protein-metal ion complex nanoparticle and the antigen peptides obtained in step S1 in water or a PBS buffer, and obtaining a nano-vaccine after incubation.

5. The preparation method according to claim 4, wherein In step S1, the concentration of the metal ions is 0.1 mol / L, and the concentration of the protein molecules is 10 mg / mL, and the surface of the protein molecules is negatively charged.

6. The preparation method according to claim 4, wherein In step S1, the reaction is a stirring reaction, the stirring temperature is 40-50°C, the stirring time is 3-5 hours, and the stirring rate is 100-1000 r / min.

7. The preparation method according to claim 4, wherein In step S2, the mass ratio of the antigen peptides to the protein-metal ion complex nanoparticles is 1:0.2-1.

8. The preparation method according to claim 4, wherein In step S2, the incubation temperature of the protein-metal ion complex nanoparticles and the antigen peptides is 25-37°C, and the incubation time is 1-3 hours.

9. Use of the antigen peptide nano-vaccine based on protein-metal ions according to any one of claims 1-3 in the preparation of a tumor vaccine.

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