Protein polyphenol nanocapsules, their preparation methods, and their application in the preparation of nanovaccines

Abstract

This invention belongs to the field of biomedical technology and relates to protein polyphenol nanocapsules, their preparation method, and their application in the preparation of nanovaccines. Eight-arm polyethylene glycol-hydroxyl groups, IMDQ, 2-MIM, and zinc salt are used in water as a solvent to prepare IMDQ-loaded ZIF-8 nanoparticles. The IMDQ-loaded ZIF-8 nanoparticles are dispersed in water, and OVA is added for mixing and reaction, allowing the IMDQ-loaded ZIF-8 nanoparticles to adsorb OVA. The OVA-adsorbed IMDQ-loaded ZIF-8 nanoparticles are then dispersed in water, and tannic acid is added for reaction, causing the tannic acid to etch ZIF-8 to form a hollow structure. This hollow structure serves as a framework for the interaction between tannic acid and OVA to form protein polyphenol nanocapsules. The protein polyphenol nanocapsules prepared by this invention exhibit good biocompatibility and immunogenicity and can be used as nanovaccines for tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to protein polyphenol nanocapsules, their preparation methods, and their application in the preparation of nanovaccines. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Compared to traditional treatments, tumor immunotherapy has fewer toxic side effects. Currently, tumor vaccines are a hot topic in tumor immunotherapy. Nanoparticle vaccines are a specific application of nanotechnology in tumor vaccine research. First, they can prevent antigens from being degraded by enzymes, thus improving their antigen stability. Second, by controlling the physicochemical properties of nanocarriers, such as size, shape, and surface chemistry, their ability to migrate to lymph nodes and their uptake capacity by antigen-presenting cells are enhanced. Finally, and more importantly, they can simultaneously deliver antigens and adjuvants to antigen-presenting cells, causing a more durable immune effect.

[0004] Although nanovaccines have made significant progress in improving immunogenicity and lymph node migration, several issues remain to be addressed. First, while antigen loading can be achieved through methods such as encapsulation, adsorption, and conjugation, these processes often involve harsh conditions such as oil-water interfaces, mechanical stirring, and high temperatures, which may damage the antigen structure and activity. Second, the complexity of preparation and low yields increase vaccine costs. Finally, the toxicity of some carriers can lead to various adverse reactions. Therefore, there is a need for a nanovaccines that are simple to prepare, highly safe, and possess enhanced immunogenicity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide protein polyphenol nanocapsules, their preparation methods, and their application in the preparation of nanovaccines. The protein polyphenol nanocapsules prepared by this invention have good biocompatibility and immunogenicity, and can be used as nanovaccines for tumor immunotherapy.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On the one hand, a method for preparing protein polyphenol nanocapsules includes the following steps:

[0008] Eight-arm polyethylene glycol-hydroxy (8-arm-PEG-OH), the immunoadjuvant imidazoquinoline (IMDQ), 2-methylimidazole (2-MIM) and zinc salt were used as a solvent to prepare zeolite imidazolate framework (ZIF-8) nanoparticles loaded with IMDQ.

[0009] IMDQ-loaded ZIF-8 nanoparticles were dispersed in water, and ovalbumin antigen (OVA) was added to carry out a mixing reaction, so that the IMDQ-loaded ZIF-8 nanoparticles adsorbed OVA.

[0010] ZIF-8 nanoparticles loaded with IMDQ and adsorbed with OVA were dispersed in water, and tannic acid was added to react with the nanoparticles. The tannic acid etched the ZIF-8 nanoparticles to form a hollow structure, and the hollow structure served as a framework to form protein polyphenol nanocapsules through the interaction of tannic acid and OVA.

[0011] This invention uses OVA as the antigen. As a protein, OVA can interact with polyphenolic compounds to form a protein-polyphenol network on a template, and after removing the template, a protein-polyphenol capsule is formed. However, studies have shown that using OVA alone as the antigen to prepare nanovaccines results in low immunogenicity and difficulty in achieving antigen-specific immune responses. Therefore, this invention adds an agonist to improve immunogenicity. Zinc ions and the adjuvant IMDQ both have agonist effects; therefore, this invention uses zinc ions to prepare metal-organic frameworks loaded with IMDQ. The loading of IMDQ can be achieved by first preparing the metal-organic framework and then adsorbing the loaded IMDQ through the porous structure of the metal-organic framework, or by adding IMDQ during the preparation of the metal-organic framework to achieve loading. However, this invention has found that both IMDQ and metal-organic frameworks (MOFs) carry positive charges, making it difficult for IMDQ to approach MOFs and resulting in low loading efficiency for IMDQ. Furthermore, MOFs require adsorption of OVA, which carries a negative charge. If MOFs are formed first and then IMDQ is loaded, the added OVA easily electrostatically binds to the IMDQ, reducing the loading efficiency of OVA. Therefore, to improve the loading efficiency of both IMDQ and OVA, this invention adds IMDQ during the MOF preparation process. By loading IMDQ into the MOF during its formation, the loading efficiency of IMDQ is improved, and the ionization of IMDQ is avoided. This prevents free IMDQ from electrostatically binding to OVA, increasing the electrostatic binding efficiency between OVA and the MOF, thus simultaneously improving the loading efficiency of both IMDQ and OVA.

[0012] Generally, the preparation of ZIF-type metal-organic frameworks using zinc ions requires organic solvents. The residual organic solvent after preparation can damage the structure and properties of proteins. This is especially true for the OVA used in this invention, which serves as both an antigen and a raw material for nanocapsules; therefore, it is necessary to avoid preparing ZIF-type metal-organic frameworks with zinc ions in organic solvents. Since IMDQ needs to be added during the preparation of the metal-organic framework, and IMDQ's steric hindrance and high nitrogen content affect the formation of ZIF-type metal-organic frameworks in water, this invention adds 8-arm-PEG-OH during the preparation of ZIF-type metal-organic frameworks in water to increase mineralization efficiency and dispersibility, thereby ensuring the formation of the ZIF-8 metal-organic framework upon the addition of IMDQ.

[0013] While zinc ions can act as immune adjuvants, excessive zinc ion release from ZIF-8 in cells can lead to high toxicity and low biocompatibility. ZIF-8 can be removed using EDTA solution, but complete removal results in a lack of scaffold support in the protein-polyphenol network, causing collapse and hindering the formation of protein-polyphenol nanocapsules. This invention uses tannic acid to etch ZIF-8 to form a hollow structure, serving as the scaffold for the protein-polyphenol nanocapsules and preventing their collapse. Furthermore, polyphenolic compounds exhibit various interactions with proteins, thus forming protein-polyphenol nanocapsules. Simultaneously, the removal of most zinc ions reverses the positive potential of ZIF-8, reducing toxicity and increasing biocompatibility.

[0014] The protein polyphenol nanocapsules prepared by this invention can simultaneously deliver OVA, the immune adjuvant IMDQ, and zinc ions, thereby greatly increasing the immunogenicity of the protein polyphenol nanocapsules.

[0015] On the other hand, a protein polyphenol nanocapsule is obtained by the above preparation method.

[0016] Thirdly, the application of the aforementioned protein polyphenol nanocapsules in the preparation of nanovaccines.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) In the process of preparing protein polyphenol nanocapsules, ZIF-8 is etched into a hollow capsule shape under the action of tannic acid after adsorbing OVA, removing most of the zinc ions and reversing the positive potential of ZIF-8, thereby improving the biocompatibility of the nanovaccine; the interaction between polyphenol and protein can further fix the protein and prevent OVA leakage; the stimulation response (pH response) of protein polyphenol capsules increases its lysosomal escape property.

[0019] (2) The protein polyphenol capsules prepared in this invention can simultaneously deliver antigen OVA, immune adjuvant IMDQ and zinc ions to the same antigen-presenting cells, which greatly enhances the immunogenicity of the nanovaccine and thus achieves an antigen-specific immune response. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 This is a transmission electron microscope image of the IMDQ@OVA-TA protein polyphenol capsules prepared in Example 4 of this invention;

[0022] Figure 2 The particle size distribution of the IMDQ@OVA-TA protein polyphenol capsules prepared in Example 4 of this invention;

[0023] Figure 3 The Zeta potential diagram is shown for the IMDQ@OVA-TA protein polyphenol capsules prepared in Example 4 of this invention.

[0024] Figure 4 Cell viability diagram of the protein polyphenol capsules prepared in Example 4 of this invention;

[0025] Figure 5 This is a cellular uptake diagram of the protein polyphenol capsules prepared in Example 4 of the present invention;

[0026] Figure 6 The effect of the protein polyphenol capsules prepared in Example 4 of this invention as a nanovaccine on the expression of CD86, a maturation marker of dendritic cells induced by mouse bone marrow mesenchymal stem cells;

[0027] Figure 7 The effect of the protein polyphenol capsules prepared in Example 4 of this invention as a nanovaccine on the expression of CD80, a maturation marker of dendritic cells induced by mouse bone marrow mesenchymal stem cells;

[0028] Figure 8 Laser confocal micrograph of the protein polyphenol capsules prepared in Example 4 of this invention after being co-cultured with RAW264.7 cells as a nanovaccine. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0031] In order to provide a nanovaccine that is simple to prepare, safe, and can improve immunogenicity, this invention proposes a protein polyphenol nanocapsule, a preparation method thereof, and its application in the preparation of nanovaccines.

[0032] A typical embodiment of the present invention provides a method for preparing protein polyphenol nanocapsules, comprising the following steps:

[0033] ZIF-8 nanoparticles loaded with IMDQ were prepared by using water as a solvent with 8-arm-PEG-OH, the immune adjuvant IMDQ, 2-MIM and zinc salt.

[0034] IMDQ-loaded ZIF-8 nanoparticles were dispersed in water, and the antigen OVA was added to carry out a mixing reaction, so that the IMDQ-loaded ZIF-8 nanoparticles adsorbed OVA.

[0035] ZIF-8 nanoparticles loaded with IMDQ and adsorbed with OVA were dispersed in water, and tannic acid was added to react with the nanoparticles. The tannic acid etched the ZIF-8 nanoparticles to form a hollow structure, and the hollow structure served as a framework to form protein polyphenol nanocapsules through the interaction of tannic acid and OVA.

[0036] The zinc salt described in this invention has a cation of zinc ions (Zn). 2+ Compounds such as zinc chloride, zinc sulfide, zinc nitrate, and zinc acetate. In some embodiments, the zinc salt is zinc nitrate. In the reaction system of the present invention, preparation using zinc nitrate yields better results.

[0037] In some embodiments, the molar ratio of zinc ions to 2-MIM is 1–3:5–10, preferably 1:3–5, and more preferably 1:3.5–4.5. Under these conditions, the material can be ensured to react as completely as possible, and the adhesion of raw materials to the pores of the generated ZIF-8 can be avoided as much as possible, thereby increasing the adsorption performance of OVA.

[0038] In some embodiments, the ratio of zinc ions to 8-arm-PEG-OH is 1–3:0.2–0.4, mmol:g, preferably 1:0.12–0.13, mmol:g. Under these conditions, the particle size of the ZIF-8 nanoparticles loaded with IMDQ can be adjusted while ensuring the formation of a metal-organic framework ZIF-8 upon the addition of IMDQ, thereby controlling the size of the protein polyphenol nanocapsules and making them more suitable for cellular uptake.

[0039] In some embodiments, the ratio of zinc ions to IMDQ is 1–3:0.1–0.2 (mmol:g), preferably 1:0.02–0.03 (mmol:g). Under these conditions, it is more conducive to the formation of ZIF-8 encapsulation of IMDQ and to reducing IMDQ loss.

[0040] In some embodiments, the temperature for preparing IMDQ-loaded ZIF-8 nanoparticles is 35–45°C, and the preparation time is 20–40 min. Under these conditions, the ZIF-8 mineralization efficiency can be further improved, which is beneficial to enhancing the stability of the IMDQ-loaded ZIF-8 nanoparticles.

[0041] In some embodiments, 8-arm-PEG-OH and IMDQ are added to water and dispersed evenly, followed by the sequential addition of 2-MIM aqueous solution and zinc salt aqueous solution. First, 8-arm-PEG-OH and IMDQ are dissolved in water to facilitate uniform dispersion of the materials, resulting in uniform product particles. Then, 2-MIM aqueous solution is added first, followed by zinc salt aqueous solution, which promotes uniform mixing of 2-MIM, zinc salt, 8-arm-PEG-OH, and IMDQ, thereby improving the loading efficiency of IMDQ and ensuring the uniformity of IMDQ loading on ZIF-8 nanoparticles.

[0042] In some embodiments, the prepared material containing ZIF-8 nanoparticles loaded with IMDQ is centrifuged and washed with water.

[0043] In some embodiments, an aqueous solution of OVA is added to the aqueous dispersion of IMDQ-loaded ZIF-8 nanoparticles. Dissolving the OVA first facilitates its dispersion, thereby improving the uniformity of OVA adsorption by the IMDQ-loaded ZIF-8 nanoparticles.

[0044] In some embodiments, the mass ratio of IMDQ-loaded ZIF-8 nanoparticles to OVA is 1–5:1–3, preferably 2–3:2. Under this condition, it is possible to ensure that the IMDQ-loaded ZIF-8 nanoparticles completely adsorb OVA, thereby improving the utilization rate of raw materials.

[0045] In some embodiments, an aqueous solution of OVA is added to an aqueous dispersion of ZIF-8 nanoparticles loaded with IMDQ, and the mixing reaction is carried out for 10 to 14 hours.

[0046] In some embodiments, an aqueous solution of tannic acid is added to an aqueous dispersion of ZIF-8 nanoparticles loaded with IMDQ and adsorbed with OVA. Pre-dissolving the tannic acid ensures its reaction efficiency and improves the uniformity of the final protein polyphenol nanocapsules, thereby enhancing the effectiveness of the protein polyphenol nanocapsules as a nanovaccine.

[0047] In some embodiments, the mass ratio of ZIF-8 nanoparticles loaded with IMDQ and adsorbing OVA to tannic acid is 1–5:1–5, preferably 2–3:2. Under these conditions, it is possible to better ensure that the tannic acid etches the ZIF-8 to form a hollow structure, and that the hollow structure serves as a framework for the interaction between tannic acid and OVA to form protein polyphenol nanocapsules.

[0048] In some embodiments, an aqueous solution of tannic acid is added to an aqueous dispersion of ZIF-8 nanoparticles loaded with IMDQ that adsorb OVA, and the reaction time is 3 to 5 hours.

[0049] In another embodiment of the present invention, a protein polyphenol nanocapsule is provided, which is obtained by the above preparation method.

[0050] In some embodiments, the average hydrated particle size is 250–350 nm.

[0051] A third embodiment of the present invention provides an application of the above-mentioned protein polyphenol nanocapsules in the preparation of nanovaccines.

[0052] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are all obtained commercially.

[0053] Example 1: Preparation of zeolite imidazole ester framework (ZIF-8) nanoparticles loaded with IMDQ.

[0054] Accurately weigh 10 mg of 8-arm-PEG-OH and 2 mg of IMDQ into 1 mL of deionized water, and sonicate to disperse them evenly. Add 1 mL of 2-MIM solution (320 mM), stir with a magnetic stirrer for 15 s, then add 2 mL of Zn(NO3)2·6H2O (40 mM). Maintain the water bath temperature at 40℃ and react for 30 min. After the reaction is complete, centrifuge at 8000 rcf for 5 min, remove the supernatant, and wash three times with deionized water to obtain IMDQ-loaded ZIF-8 nanoparticles, denoted as IMDQ@ZIF.

[0055] Using the same method as in this embodiment, ZIF-8 nanoparticles were obtained without adding IMDQ during the preparation process.

[0056] Example 2: FITC-labeled model antigen ovalbumin OVA.

[0057] Accurately weigh 1 mg of FITC and dissolve it in 30 μL of dimethyl sulfoxide (DMSO). Store in the dark.

[0058] Accurately weigh 30 mg of ovalbumin (OVA), dissolve it in 5 mL of deionized water, and sonicate it to dissolve and disperse.

[0059] Mix the prepared FITC solution with the OVA solution and stir magnetically for 2 hours. Place the mixture in a dialysis bag at 3500K and dialyze for 48 hours using sodium carbonate-sodium bicarbonate buffer solution (pH = 9-10), changing the water every 6 hours. After dialysis, collect the sample in a centrifuge tube and freeze-dry for 24 hours to obtain the FITC-labeled OVA sample.

[0060] Example 3: ZIF-8 nanoparticles loaded with IMDQ adsorb antigen OVA.

[0061] The nanoparticles (ZIF-8 nanoparticles loaded with IMDQ) obtained in Example 1 were dispersed in water, and the particle concentration was controlled at 2.5 mg / mL.

[0062] Accurately weigh the antigen OVA, dissolve it in deionized water, and control the concentration of OVA to 2 mg / mL. Vortex for 15 seconds to ensure complete dissolution.

[0063] An equal volume of the antigen OVA solution was added to the nanoparticle solution, and the mixture was vortexed for 15 seconds to ensure thorough mixing. The mixture was then stirred for 12 hours. After the reaction, the nanoparticles were centrifuged at 8000 rcf for 5 minutes to remove the supernatant. The nanoparticles were then washed three times with deionized water to obtain ZIF-8 nanoparticles loaded with IMDQ and adsorbed with OVA, denoted as IM.@ZIF@OVA.

[0064] The same method used in this embodiment can be used to prepare ZIF-8 nanoparticles loaded with the immune adjuvant IMDQ and adsorbed with FITC-labeled antigen OVA.

[0065] Using the same method as in this embodiment, nanoparticles in which ZIF-8 adsorbs antigen OVA and FITC-labeled antigen OVA can be prepared respectively.

[0066] Example 4: Preparation of protein polyphenol capsules loaded with the immune adjuvant IMDQ.

[0067] The nanoparticles (ZIF-8 nanoparticles loaded with IMDQ and adsorbed with OVA) obtained in Example 3 were dispersed in water, and the particle concentration was controlled at 2.5 mg / mL.

[0068] Accurately weigh 40 mg of tannic acid, dissolve it in 1 mL of deionized water, and sonicate it to disperse it evenly.

[0069] Add 50 μL of tannic acid solution to 1 mL of nanoparticle solution, vortex for 15 s to ensure thorough mixing, then stir for 4 h. After the reaction is complete, centrifuge at 8000 rcf for 5 min, remove the supernatant, and wash three times with deionized water to obtain protein polyphenol capsules, i.e., IMDQ@OVA-TA. The obtained protein polyphenol capsules are stored at 4 °C.

[0070] Using the same method as in this embodiment, replacing the ZIF-8 nanoparticles loaded with IMDQ that adsorb OVA with ZIF-8 nanoparticles that adsorb OVA, protein polyphenol capsules of ZIF-8@OVA-TA can be prepared.

[0071] The morphology of the IMDQ@OVA-TA protein polyphenol capsules prepared in Example 4 is as follows: Figure 1 As shown, this demonstrates that the IMDQ@OVA-TA prepared in Example 4 has a capsule morphology.

[0072] The particle size distribution of the IMDQ@OVA-TA protein polyphenol capsules prepared in Example 4 is as follows: Figure 2 As shown, this indicates that its average hydrated particle size is about 300 nm.

[0073] The zeta potentials of IMDQ@ZIF prepared in Example 1, IM.@ZIF@OVA prepared in Example 3, and IMDQ@OVA-TA prepared in Example 4 are as follows: Figure 3 As shown, the zeta potential can be reversed after adsorption of OVA, and the negative potential of the nanoparticles can be further increased under the action of tannic acid.

[0074] Application examples:

[0075] RAW264.7 cell culture: Preheat DMEM medium to 37°C in a water bath. Transfer 5 mL of preheated medium to a 15 mL centrifuge tube in a biosafety cabinet. Remove RAW264.7 cells from the liquid nitrogen container and quickly place them in a 37°C water bath, gently shaking to ensure even heating until thawed. Transfer the thawed cell suspension to a pre-separated medium in the biosafety cabinet, centrifuge, and discard the supernatant. Resuspend the cells in medium, transfer to a culture flask, mix well, and incubate in an incubator. When the cells reach 80% confluence, scrape them off with a cell scraper, transfer to a centrifuge tube, centrifuge, and discard the supernatant. Resuspend the cells in 1 mL of medium, transfer 250 μL of the cell suspension to a culture flask, add the required amount of medium, mix well, and incubate in an incubator.

[0076] The cell viability assay for the nanovaccine: At the third passage, newly revived cells were counted based on cell counts at a ratio of 2 × 10⁶ cells per well. 4 Cells were seeded into 96-well plates at a density of 100 μL of culture medium per well. After seeding, the plates were shaken well and incubated overnight. The next day, ZIF-8, ZIF-8@OVA-TA, and IMDQ@OVA-TA nanomaterials were prepared at concentrations of 10, 20, 40, 60, 80, and 100 μg / mL using culture medium. The original culture medium in the 96-well plates was removed, and fresh medium containing different concentrations of nanomaterials was added. A control group without the nanomaterials was used. Six replicates were made for each group. After adding the nanomaterials, the plates were incubated for 24 hours. After 24 hours, the supernatant was aspirated, and each well was washed three times with PBS. 100 μL of prepared MTT solution (0.5 mg / mL) was added under light-protected conditions, and the plates were incubated for 4 hours. Under light-protected conditions, the supernatant was aspirated, and 100 μL of DMSO was added to each well. The plates were shaken for 20 minutes. The absorbance at 490 nm was measured using a multi-mode microplate reader. Cell viability was calculated using a formula. The results are as follows: Figure 4 As shown, the prepared protein polyphenol capsules can reduce the toxicity of ZIF-8 and have good biocompatibility.

[0077] The nanovaccine cell uptake assay was performed as follows: During RAW264.7 cell passage, based on cell count results, cells were added at a rate of 2.5 × 10⁶ cells per well. 5Cells were seeded at a density of 0.5 mL of culture medium per well into 24-well plates, ensuring even distribution during seeding. The plates were then incubated overnight. The next day, pre-prepared FITC-labeled OVA and IMDQ@OVA-TA were added to the culture medium (OVA concentration controlled at 20 μg / mL). The supernatant was aspirated, and 1 mL of culture medium containing different materials was added to each well. All procedures were performed in the dark. Four replicates were made for each material, with a PBS group reserved as a negative control. After drug addition, the plates were incubated for 8 hours. After this time, the supernatant was discarded, and the cells were washed three times with PBS. Cells were then collected for flow cytometry analysis. Results are as follows: Figure 5 As shown, after 8 hours of incubation with PBS and OVA, the degree of cell internalization was 0.05% and 0.265%, respectively. However, after 8 hours of incubation with IMDQ@OVA-TA, the degree of internalization reached 77.455%, significantly increasing the cell uptake. This demonstrates that the prepared protein polyphenol capsules can significantly increase cell uptake compared to free antigen OVA, thereby effectively improving the efficacy of protein polyphenol capsules as nanovaccines.

[0078] Isolation and induction of mouse bone marrow cells: 4-6 week old C57BL / 6 mice were euthanized by cervical dislocation. The mice were immersed in 75% ethanol for 2 minutes, then fixed in a foam box. The tibia and femur were harvested, and the attached muscles were removed. The tibia and femur were then immersed in 75% ethanol for 10 seconds and placed in 1640 medium. Holes were made at both ends of the bone. Using a 1 mL sterile syringe, the medium was drawn and inserted into the bone marrow cavity from one end of the bone. The tibia and femur were repeatedly flushed 3-4 times until the bone shaft turned white. The bone marrow flushing solution was placed in a 50 mL sterile centrifuge tube to obtain the bone marrow cell mixture. All the above operations required a sterile environment and were performed on ice to maintain cell viability. Centrifuge the bone marrow cell mixture (1600 rcf, 5 min), discard the supernatant, add 1 mL of erythrocyte lysis buffer, mix thoroughly by pipetting, let stand for about 5 min, add 1640 medium to stop lysis, centrifuge again, discard the supernatant, add 1640 medium again, resuspend the cells, repeat twice, finally add 6 mL of 1640 complete culture medium (containing 20 ng / mL GM-CSF and 10 ng / mL IL-4) to resuspend the cells, and mix well. Seed 1 mL of the obtained cells into each well of a 6-well cell culture plate, and add 2 mL of 1640 medium to each well. Incubate in an incubator, marking it as day 0; on day 2, carefully aspirate 1.5 mL of culture medium and add fresh culture medium, then change half the medium daily thereafter, and culture until day 6 to obtain dendritic cells induced by bone marrow mesenchymal stem cells.

[0079] The effect of the nanovaccine on the activation of bone marrow-derived dendritic cells: First, dendritic cells induced by GM-CSF for 6 days were gently pipetted and collected into 50 mL centrifuge tubes, centrifuged (1600 rcf, 5 min), and resuspended in 1640 whole culture medium; 10 μL of cells were diluted with 90 μL of 1640 whole culture medium and then dropped onto a cell counting plate for counting, at a seeding density of 5.0 × 10⁶ cells / mL. 5 The cells obtained above were seeded into 24-well cell culture plates and cultured overnight in an incubator. Then, culture media solutions of OVA, OVA&IMDQ, ZIF-8@TA (denoted as ZT), ZIF-8@OVA-TA (denoted as ZOT), and IMDQ@OVA-TA (denoted as IZOT) were prepared in advance, with the OVA concentration controlled at 10 μg / mL and the IMDQ concentration at 2 μg / mL. The original culture medium was gently aspirated, and fresh culture medium containing different formulations was added to co-culture the dendritic cells for 24 h, with 3 replicates per group. After incubation, the cells were forcefully blown off and transferred to 1.5 mL centrifuge tubes, centrifuged (1600 rcf, 5 min), and the supernatant was stored for later experiments. PBS containing APC-CD11c antibody, PE-Cy7-CD86 antibody, and FITC-CD80 antibody were prepared in advance. Add 100 μL of antibody-containing PBS to each centrifuge tube, reserving for Blank and single-stained samples. Incubate at 4°C in the dark. After 30 min, wash cells with 1 mL of PBS, resuspend cells in PBS, and perform flow cytometry analysis. The activation of bone marrow-derived dendritic cells (BMDCs) by nanoparticles was analyzed by the fluorescence intensity and percentage shown in the flow cytometer. BMDC maturation is a key step in initiating the immune response, accompanied by upregulation of co-stimulatory markers (CD80, CD86). Results are as follows: Figure 6 and Figure 7 As shown, compared with the PBS group, although the free drug (OVA, OVA & IMDQ), ZT, and ZOT groups could upregulate the expression of CD80 and CD86 to some extent, their expression levels were still lower than those promoted by IZOT in BMDCs. This can be attributed to the lower cellular uptake of the free drug, the lower immunogenicity of zinc ions themselves, and the lack of activation effect from the immune adjuvant. The prepared protein polyphenol capsule nanovaccine (IZOT) not only improved antigen uptake but also achieved co-delivery of antigen and adjuvant, thus significantly increasing the expression levels of the maturation markers CD86 and CD80 on the surface of BMDCs. This demonstrates the superiority of the prepared nanovaccine in promoting BMDC maturation and its potential to stimulate antigen-specific immune responses.

[0080] The lysosomal escape function of the nanovaccine was detected as follows: During passage of RAW264.7 cells, based on the cell count results, 1 × 10⁶ cells were collected per well. 6Cells were seeded at a density of cells / well into laser confocal microscopy culture dishes and incubated overnight. Then, pre-prepared FITC-labeled OVA and IMDQ@OVA-TA culture media were added, maintaining the OVA concentration at 20 μg / mL, and the cells were cultured for another 8 hours. After culture, the supernatant was discarded, and the cells were washed three times with PBS. Then, 0.5 mL of 4% paraformaldehyde was added, and the cells were fixed in an incubator for 20 min. After the fixation time, the supernatant was discarded, and the cells were washed three times with PBS. 500 μL of Lyso-Tracker Red dye (50 nM) was added, and the cells were incubated for 120 min to stain lysosomes. After staining, the cells were washed three times with PBS. Then, 500 μL of Hoechst 33342 nuclear dye (0.5 μg / mL) was added, and the cells were incubated for 20 min. The cells were washed three times with PBS, and finally, 0.5 mL of PBS was added. The cells were observed and photographed using a laser confocal microscope. Results are as follows: Figure 8 As shown, after treating cells with FITC-OVA alone, green fluorescence was distributed around the cell nucleus, and this green fluorescence overlapped with the red fluorescence of the labeled lysosomes, indicating that OVA was mainly located within lysosomes. In contrast, after treating cells with IMDQ@OVA-TA, free green fluorescence was distributed in the cytoplasm, and the green fluorescence did not completely overlap with the red fluorescence of the labeled lysosomal membrane, indicating that some particles escaped from the lysosomes. Antigen escape from lysosomes may facilitate cross-presentation of antigens by antigen-presenting cells, thereby inducing strong cellular immunity.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing protein polyphenol nanocapsules, characterized by, The method comprises the following steps: 8-arm-PEG-OH and IMDQ are dispersed in water, and then 2-MIM aqueous solution and zinc salt aqueous solution are added in sequence; the temperature for preparing the IMDQ-loaded ZIF-8 nanoparticles is 35-45 ℃, and the preparation time is 20-40 min; The IMDQ-loaded ZIF-8 nanoparticles are dispersed in water, and antigen OVA is added and mixed for 10-14 h to enable the IMDQ-loaded ZIF-8 nanoparticles to adsorb OVA; The OVA-adsorbed IMDQ-loaded ZIF-8 nanoparticles are dispersed in water, and tannic acid is added and reacted for 3-5 h to enable tannic acid to etch ZIF-8 to form a hollow structure, and the hollow structure as a skeleton interacts with OVA through tannic acid to form protein polyphenol nanocapsules; The zinc salt is zinc nitrate; The molar ratio of zinc ions to 2-MIM is 1:3.5-4.5; The addition ratio of zinc ions to 8-arm-PEG-OH is 1:0.12-0.13, mmol:g; The addition ratio of zinc ions to IMDQ is 1:0.02-0.03, mmol:g; The mass ratio of the IMDQ-loaded ZIF-8 nanoparticles to OVA is 2-3:2; The addition mass ratio of the OVA-adsorbed IMDQ-loaded ZIF-8 nanoparticles to tannic acid is 2-3:

2.

2. The method for preparing protein polyphenol nanocapsules as described in claim 1, characterized in that, Tannic acid aqueous solution is added to the water dispersion of the OVA-adsorbed IMDQ-loaded ZIF-8 nanoparticles.

3. A protein polyphenol nanocapsule characterized by, The protein polyphenol nanocapsules are prepared by the method of any one of claims 1-2.

4. The protein polyphenol nanocapsule as described in claim 3, characterized in that, The average water and particle size is 250-350 nm.

5. Use of the protein polyphenol nanocapsules of claim 3 or 4 in the preparation of a nano-vaccine.

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