OVA (at) MMSNs (at) BM-Mannose bionic nano vaccine and preparation method thereof

Abstract

The invention discloses an OVA (at) MMSNs (at) BM-Mannose bionic nano vaccine and a preparation method thereof, and belongs to the technical field of nano vaccines. The OVA (at) MMSNs (at) BM-Mann rose bionic nano vaccine is a nano material coated by a bacterial plasma membrane. The OVA (at) MMSNs (at) BM-Mannose bionic nano vaccine prepared by the preparation method disclosed by the invention is strong in cellular uptake ability and good in storage stability; the hemolytic activity is low, and the hemolysis rate of 6% or below can still be kept even when the concentration is 100 micrograms per milliliter; the in-vitro cytotoxicity is small, and the safety is high; obvious toxicity is not generated on mice in vivo; the hydration particle size is 220nm; dC2.4 cells have the phenomena of continuous uptake and concentration dependence on the nano vaccine, are basically saturated within 2 hours, and have the potential of being applied to the field of magnetic resonance imaging; the activation efficiency on DC2.4 is high; the targeting delivery capability is realized; according to the prepared FITC-OVA (at) MMS Ns (at) BM-Mannose bionic nano vaccine, the visual delivery of the nano vaccine can be realized, and the observation and research are convenient; the preparation method of the bionic nano vaccine is simple and easy to operate, and the used raw materials are safe, non-toxic or low in toxicity and safe to the environment and operators.

Description

Technical Field

[0001] The present invention relates to the technical field of nano vaccines, and in particular to an OVA@MMSNs@BM-Mannose bionic nano vaccine and a preparation method thereof. Background Art

[0002] In the past 30 years, the incidence of cutaneous malignant melanoma (CMM) has continued to rise steadily, and its growth rate has almost exceeded that of most malignant tumors. Since 2004, the incidence of melanoma has increased by 3% per year. At present, the clinical treatment of CMM is mainly surgical resection and radiotherapy and chemotherapy. However, traditional radiotherapy and chemotherapy can cause damage to normal cells during the treatment process and have many side effects. Therefore, finding an effective treatment for CMM is of great significance to clinical medicine.

[0003] With the advancement and development of science and technology, tumor immunotherapy, as a new treatment method, has become an important means in the clinical treatment of malignant tumors. Anti-tumor immunotherapy inhibits or eliminates tumor cells by acting on the human immune system, without side effects such as drug resistance, local treatment damage, and damage to the human immune system. Tumor vaccines can induce effective anti-tumor immunity and long-term immune memory by delivering tumor antigens to antigen-presenting cells (APCs) to activate tumor-specific immune responses, thereby inhibiting tumor growth, recurrence, and metastasis. In this process, antigens and adjuvants are key components to induce immune responses and enhance their effects. However, due to the problems of low inherent lymphatic drainage efficiency and immune-related adverse reactions caused by distribution in non-tumor sites, the clinical transformation of tumor vaccines remains challenging. Therefore, it is crucial to develop an effective delivery system to achieve efficient and targeted delivery of antigens and adjuvants.

[0004] Tumor vaccines based on nanomaterials (i.e., nanovaccines) can effectively solve the efficiency problem of co-delivery of antigens and adjuvants. Nanovaccines are prepared by electrostatic interactions, hydrophobic interactions, and covalent linkages to achieve co-delivery of antigens and adjuvants, and can specifically target APCs, induce antigen cross-presentation, or interact with other cells such as macrophages in lymphoid organs, thereby enhancing anti-tumor cellular immunity and humoral immune responses. Among them, mesoporous silica (MSNs) nanoparticles have many advantages such as large specific surface area, porous structure, good biocompatibility, and surface modifiability, and have received widespread attention in biomedical applications. As a therapeutic drug carrier, MSNs can effectively control drug release. However, due to the relatively stable Si-O-Si skeleton inside, it is relatively difficult to degrade under physiological conditions, which can easily lead to accumulation in the body and pose a potential biosafety risk. Summary of the invention

[0005] The purpose of the present invention is to provide an OVA@MMSNs@BM-Mannose bionic nanovaccine and a preparation method thereof, so as to provide a nanovaccine against CMM and solve the problem that MSNs are difficult to degrade under physiological conditions, easily accumulate in the body, and have potential safety risks.

[0006] To achieve the above object, the present invention provides an OVA@MMSNs@BM-Mannose bionic nanovaccine, wherein the OVA@MMSNs@BM-Mannose bionic nanovaccine is a nanomaterial coated with a bacterial plasma membrane.

[0007] Preferably, the nanomaterial is metal-doped mesoporous silica nanoparticles loaded with ovalbumin.

[0008] Preferably, the metal-doped mesoporous silica nanoparticles are manganese-doped mesoporous silica nanoparticles.

[0009] A visualized FITC-OVA@MMSNs@BM-Mannose biomimetic nanovaccine is prepared by modifying fluorescein isothiocyanate on ovalbumin used in the above-mentioned OVA@MMSNs@BM-Mannose biomimetic nanovaccine.

[0010] A method for preparing the OVA@MMSNs@BM-Mannose bionic nanovaccine as described above comprises the following steps:

[0011] S1. Preparation of mesoporous silica nanoparticles, MSNs;

[0012] S2, preparation of manganese-doped mesoporous silica nanoparticles, MMSNs;

[0013] S3, loading ovalbumin onto the prepared manganese-doped mesoporous silica nanoparticles to obtain ovalbumin-loaded MMSNs, OVA@MMSNs;

[0014] S4, preparation of bacterial plasma membrane, BM;

[0015] S5, coating BM with OVA@MMSNs to obtain OVA@MMSNs@BM;

[0016] S6. Preparation of OVA@MMSNs@BM-Mannose bionic nanovaccine.

[0017] Preferably, the preparation method of OVA@MMSNs in step S3 is: dissolving ovalbumin with ddH2O, adding MMSNs powder, ultrasonicating for 10 minutes, reacting at room temperature for 24 hours under stirring conditions, collecting the precipitate by centrifugation, and washing with ddH2O for 3 times.

[0018] Preferably, the mass volume ratio of ovalbumin:ddH2O:MMSNs is 40 mg:1 mL:4 mg.

[0019] Preferably, in step S5, OVA@MMSNs@BM is prepared by mechanical co-extrusion, specifically, BM and OVA@MMSNs are mixed in a mass ratio of 1:1, ultrasonicated for 20 minutes, and then passed through 400nm and 200nm polycarbonate membranes, respectively, and extruded back and forth 15-20 times.

[0020] Preferably, the method for preparing the OVA@MMSNs@BM-Mannose biomimetic nanovaccine in step S6 is: dissolving mannose molecules in ddH2O, then adding OVA@MMSNs@BM to react, and centrifuging and washing after the reaction is completed.

[0021] Preferably, the mass volume ratio of mannose:ddH2O:OVA@MMSNs@BM is 1 mg:1 mL:10 mg; after adding OVA@MMSNs@BM, the mixture is reacted at 37°C for 4 hours.

[0022] Therefore, the present invention provides an OVA@MMSNs@BM-Mannose bionic nano vaccine and a preparation method thereof, and its specific technical effects are as follows:

[0023] (1) The OVA@MMSNs@BM-Mannose bionic nanovaccine prepared by the present invention has strong cell uptake ability and good storage stability. It does not aggregate after being stored at 4°C for 7 days. It has low hemolysis, and even at a concentration of 100 μg / mL, it can still maintain a hemolysis rate of less than 6%. It has low cytotoxicity. Within the concentration range of 0-100 μg / mL, the activity of DC2.4 cells is ≥85%, and it has high in vitro safety. In vivo experiments show that it has no obvious toxicity to mice.

[0024] (2) The OVA@MMSNs@BM-Mannose bionic nanovaccine prepared by the present invention can disintegrate in an acidic environment by utilizing the pH-responsive characteristics of MMSNs, accelerating the release of the internally loaded drug. 2+ Can be used for magnetic resonance imaging;

[0025] (2) The FITC-OVA@MMSNs@BM-Mannose bionic nanovaccine prepared by the present invention can realize the visual delivery of nanovaccine, which is convenient for observation and research;

[0026] (3) The OVA@MMSNs@BM-Mannose bionic nanovaccine prepared by the present invention has a hydrated particle size of 220 nm; DC2.4 cells have a continuous uptake and concentration-dependent phenomenon for the nanovaccine, which basically reaches saturation at 2 hours, and has the potential for application in the field of magnetic resonance imaging; the activation efficiency of DC2.4 is high; and it has targeted delivery capabilities;

[0027] (4) The bionic nano-vaccine preparation method provided by the present invention is simple and easy to operate. The raw materials used are safe and non-toxic or have low toxicity, and are safe for the environment and operators. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0029] Figure 1 It is the magnetic resonance imaging T1WI image and pseudo-color image of MMSNs under the conditions of pH=7.4 and pH=5.0 in Example 1 of the present invention;

[0030] Figure 2 TEM images of multiple MMSNs (A) and OVA@MMSNs@BM-Mannose (C) and single MMSNs (B) and OVA@MMSNs@BM-Mannose (D) in Example 1 of the present invention;

[0031] Figure 3 It is the characterization of the hydrated particle size and surface potential of different nanoparticles in Example 1 of the present invention, (A) particle size distribution diagram of MMSNs, OVA@MMSNs, OVA@MMSNs@BM, OVA@MMSNs@BM-Mannose; (B) Zeta potential change diagram of MMSNs, OVA@MMSNs, OVA@MMSNs@BM, OVA@MMSNs@BM-Mannose;

[0032] Figure 4 It is the fluorescence spectra of FITC-OVA, MMSNs, and FITC-OVA@MMS Ns@BM-Mannose in Example 2 of the present invention;

[0033] Figure 5 This is the curve of the change of the hydrated particle size and PDI of OVA@MMSNs@BM-Mannose over time under the storage condition of 4°C in Example 3 of the present invention;

[0034] Figure 6The in vitro safety test results of the OVA@MMSNs@BM-Mannose nanovaccine in Example 4 of the present invention are shown in Figure 1. Part A shows the results of the hemolysis test of red blood cells of the OVA@MMSNs@BM-Mannose nanovaccine. Part B shows the results of the cytotoxicity test of the OVA@MMSNs@BM-Mannose nanovaccine on DC2.4 cells.

[0035] Figure 7 The quantitative analysis of the uptake of DC2.4 cells co-incubated with FITC-OVA@MMSNs@BM-Mannose nanovaccine at 0h, 1h, 2h, and 4h by flow cytometry in Example 5 of the present invention (A); Part B is a quantitative analysis of the proportion of FITC-positive DC2.4 cells; compared with the 0h group, *P<0.05, **P<0.01;

[0036] Figure 8 This is the observation of the uptake of FITC-OVA@MMSNs@BM-Mannose nanovaccine by DC2.4 cells after co-incubation with the nanovaccine for 0h, 0.5h, 1h, and 2h under a confocal microscope in Example 5 of the present invention; scale: 35μm;

[0037] Fig. 9 It is the MRI image T1WI and pseudo-color image after DC2.4 cells were co-incubated with different concentrations of OVA@MMSNs@BM-Mannose in Example 6 of the present invention;

[0038] Fig.10 This is the analysis result of cell activation after DC2.4 cells were co-incubated with different materials in Example 7 of the present invention;

[0039] Fig.11 The ELISA kit in Example 8 of the present invention was used to analyze the secretion of IFN-β after DC2.4 cells were co-incubated with different materials; compared with the control group, *P<0.05, **P<0.01;

[0040] Fig.12 The results of blood routine test (A) and blood biochemistry (B) of mice injected with PBS and OVA@MMSNs@BM-Manno se nanovaccine in Example 9 of the present invention;

[0041] Fig.13 This is a histopathological image of the main organs (heart, liver, spleen, lung, and kidney) of mice after injection of OVA@MMSNs@BM-Mannose nanovaccine in Example 10 of the present invention. Scale: 200 μm. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0043] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0044] The instruments, equipment, reagents and materials used in the examples were obtained through commercial channels, and the methods and steps not described in detail in the examples are well known in the art.

[0045] Embodiment 1

[0046] Preparation of OVA@MMSNs@BM-Mannose bionic nanovaccine, the specific steps are as follows:

[0047] 1) Synthesis of mesoporous silica nanoparticles (MSNs). Weigh 150 mg of triethanolamine (TEA) with an electronic balance, place it in a round-bottom flask, add 46 mL of deionized water (ddH2O), place the round-bottom flask on a magnetic stirrer, set the conditions to 500 rpm, 5 min, and fully dissolve it. Slowly drop 10 mL of a pre-configured 25% hexadecyltrimethylammonium chloride (CTAC) solution into the TEA solution. Under oil bath conditions, set the speed to 400 rpm and the temperature to 60 ° C. After reacting for 1.5 hours, adjust the speed to 120 rpm, and slowly drop 10 mL of a mixed solution of cyclohexane and tetraethoxysilane (TEOS) (volume ratio of 19:1) and react for 12 hours. After the reaction is completed, centrifuge at 16000 rpm for 30 minutes to collect the white precipitate, i.e., mesoporous silica nanoparticles (MSNs).

[0048] 2) Preparation of MMSNs. Pipette 20 mL of 10 mM hexadecyltrimethylammonium bromide (CTAB) solution, add the MSNs obtained above, set the speed to 600 rpm under oil bath conditions, add 2 mL of anhydrous ethanol and 1.24 g of TEA one by one, and mix them thoroughly. After 10 min, add 0.6 mL of manganese sulfate solution (8 mg / mL) to the reaction system, and set the temperature to 60 ° C. When the reaction system is heated to 60 ° C, add 90 μL of a mixed solution of TEOS and 3-aminopropyltriethoxysilane (APTES) dropwise (volume ratio of 10:1). After reacting for 12 h, centrifuge at 12000 rpm for 30 min to collect the brown precipitate. Wash with ddH2O and ethanol several times (centrifugation conditions set to 12000 rpm, 30 min) to remove residual reactants and CTAB surfactant. The collected brown precipitate is reacted with 1% (wt) sodium chloride-methanol solution at 37 ° C for 12 h, and repeated three times. Finally, the sample was vacuum dried at 40 °C for 24 h to obtain a brown precipitate, which was manganese-doped mesoporous silica nanoparticles (MMSNs).

[0049] MMSNs were dissolved in 10 mL of ddH2O to obtain MMSNs solution, 100 μL of the solution was mixed with 200 μL of concentrated nitric acid solution, placed in an oven at 80°C for 0.5 h, 1.7 mL of ddH2O was added, and the manganese ion concentration in the solution was detected by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the manganese ion concentration was 35.44 mg / L.

[0050] According to the results of manganese ion concentration detection, different concentrations of MMSNs solutions (0-0.64mM) were prepared under the conditions of pH=7.4 and pH=5, and MRI scanning was performed. The scanned images were uploaded to the GE AW4.6 processing workstation to obtain T1WI images and T1map images of MMSNs. The MRI scanning parameters were: TR=3000ms, TE=10ms, inversion time=200-800ms, matrix=384×224, FOV=18cm×18cm, slice thickness=3.0mm, and slice distance=1.5mm.

[0051] T1WI images of MMSNs are as follows Figure 1 As shown, with the increase of Mn 2+ As the concentration increases, the T1WI signal value of the ROI area increases, and at the same time, the pseudo-color image gradually changes from blue to red. This confirms that the MRI T1WI signal value of MMS Ns is concentration-dependent. 2+ The T1WI signal value of MMSNs in the slightly acidic environment (pH = 5.0) was higher than that in the normal environment (pH = 7.4), which was mainly due to the degradation of MMSNs in the slightly acidic environment, releasing more Mn2+ , which can be used for MRI imaging.

[0052] 3) Preparation of MMSNs nanoparticles loaded with ovalbumin (OVA) (OVA@MMS Ns). Weigh 200 mg of ovalbumin (OVA) using an electronic balance and dissolve it in 5 mL of ddH2O to obtain an OVA solution with a concentration of 40 mg / mL. Weigh 20 mg of MMSNs powder and add it to the OVA solution. After ultrasonic treatment for 10 min, place it on a magnetic stirrer (250 rpm) and react at room temperature for 24 h. After the reaction is completed, centrifuge at 12000 rpm for 30 min and wash 3 times with ddH2O to obtain OVA@MMSNs.

[0053] 4) Extraction of bacterial plasma membrane (BM). Cryopreserved E. coli DH5α cells were cultured in liquid culture medium at 37°C and shaken at 200 rpm overnight. 600 When it reaches 1.2, centrifuge at 3000g for 20min at 4°C, collect the bacteria, and wash 3 times with PBS. Resuspend the bacterial pellet in 10mL buffer A (containing 1M sucrose, 0.2M Tris-HCl, pH=8.0). Then add lysozyme to a final concentration of 2mg / mL. Incubate the bacteria at 120rpm and 37°C for 1h. Then add 90mL sterile water containing DNase (10μg / mL) and mix gently. Collect the pellet by centrifugation at 3000g for 20min at 4°C, and resuspend it in 10mL buffer B (containing 20mM Tris-HCl, pH=7.2, 50mMNaCl, 5mM EDTA) containing 20% ​​(w / v) sucrose for cell lysis. The supernatant was collected and placed on top of a discontinuous sucrose gradient (from bottom to top: 10 mL 50% sucrose, 5 mL 46% sucrose, 10 mL 42% sucrose, 10 mL 36% sucrose, 5 mL 32% sucrose, and 10 mL 27% sucrose) and centrifuged at 113,000 g for 2 h at 4 ° C using an ultracentrifuge. Subsequently, 1.5 mL of the cytoplasmic membrane-containing fraction was extracted and diluted with ice-cold buffer B to reduce the sucrose concentration to 10% (w / v). The bacterial plasma membrane (BM) was collected by centrifugation at 113,000 g for 1 h at 4 ° C, resuspended in PBS, and stored at -80 ° C for later use.

[0054] 5) BM-coated OVA@MMSNs (OVA@MM SNs@BM) were prepared by mechanical co-extrusion. The collected BM was mixed with OVA@MMSNs at a mass ratio of 1:1, and the mixed solution was placed in an ultrasonicator and ultrasonicated for 20 min at a frequency of 42 kHz and a power of 100 W. Then, the mixed solution was passed through a 400 nm and 200 nm polycarbonate membrane using a liposome extruder, and extruded back and forth 20 times to obtain OVA@MMSNs@BM, which was stored at 4°C for future use.

[0055] 6) Preparation of manganese-doped mesoporous silica biomimetic nanovaccine (OVA@MMSNs@BM-Mannose). Use an electronic balance to weigh 1 mg of mannose molecules (DSPE-PEG-Mannos e) and dissolve it in 1 mL of ddH2O. Take 10 mL of the above-prepared OVA@MMSNs@BM solution (1 mg / mL), add DSPE-PEG-Mannose solution, and react at 37°C for 4 hours. After the reaction is completed, centrifuge at 12000 rpm for 30 minutes, wash 3 times with ddH2O to remove the unreacted DSPE-PEG-Mannose. The obtained precipitate is the manganese-doped mesoporous silica biomimetic nanovaccine (OVA@MMSNs@BM-Mannose).

[0056] The prepared MMSNs and OVA@MMSNs@BM-Mannose were observed by TEM. Figure 2 As shown in the figure, MMSNs showed a spherical structure under electron microscopy, with good monodispersity, uniform size, and obvious mesoporous structure. The OVA@MMSNs@BM-Mannose image showed that the surface of MMSNs was wrapped with a layer of cell membrane structure, indicating that BM had been successfully coated on the surface of MMSNs.

[0057] The particle size and Zeta potential of the prepared MMSNs, OVA@MMSNs, OVA@MMSNs@BM, and OVA@MMSNs@BM-Mannose were analyzed. Figure 3 As shown in the figure, the particle sizes of the prepared MMSNs, OVA@MMSNs, OVA@MMSNs@BM and OVA@MMS Ns@BM-Mannose were approximately 164, 164, 190 and 220 nm. After coating with bacterial membrane and inserting mannose molecules, the hydrated particle size of the nanoparticles increased slightly, but remained at about 200 nm.

[0058] Embodiment 2

[0059] The steps for preparing FITC-OVA@MMSNs@BM-Mannose biomimetic nanovaccine are the same as those in Example 1, except that in step 3), OVA is first modified with fluorescein isothiocyanate (FITC) to obtain FITC-modified OVA, FITC-OVA, and then FITC-OVA@MMSNs is prepared by the method of step 3).

[0060] Then, according to steps 4) to 6) in Example 1, except that FITC-OVA@MMSNs was substituted for OVA@MMSNs in the steps of Example 1, to prepare FITC-OVA@MMSNs@BM-Mannose.

[0061] The fluorescence spectrum of FITC-OVA@MMSNs@BM-Mannose is shown in the figure Figure 4 As shown, MMSNs do not have a characteristic fluorescence absorption peak at 520 nm, while FITC-OVA and FITC-OVA@MMSNs@BM-Mannose both have characteristic fluorescence absorption peaks at 520 nm, indicating that FITC-OVA has been successfully loaded into MMSNs and the visualized delivery of nanovaccines can be achieved.

[0062] Embodiment 3

[0063] The storage stability of the OVA@MMSNs@BM-Mannose bionic nanovaccine prepared in Example 1 was investigated, and the specific steps were as follows:

[0064] The OVA@MMSNs@BM-Mannose solution was placed in a 4°C refrigerator, and the changes in its hydrodynamic diameter (Dh) and polydispersity index (PDI) were measured using dynamic light scattering (DLS) on the 1st, 3rd, 5th, and 7th days.

[0065] The results are as follows Figure 5 As shown in the figure, the hydrated particle size of OVA@MMSNs@BM-Mannose is relatively constant, and the PDI coefficient is less than 0.5. At the same time, the solution is always in a clear and transparent state without obvious precipitation, indicating that OVA@MMSNs@BM-Mannose is not easy to agglomerate at 4°C and is easy to store.

[0066] Embodiment 4

[0067] The in vitro safety of the OVA@MMSNs@BM-Mannose nanovaccine prepared in Example 1 was investigated, and the specific steps were as follows:

[0068] 1) Collect mouse blood by eyeball blood sampling, dissolve with saline and centrifuge, collect cell pellets, and wash with saline three times. Use a pipette to draw 200 μL of red blood cells, add 10 mL of saline solution to dilute, mix well, and take 500 μL of red blood cell solution and add it to 1.5 mL centrifuge tubes. Add 6.25 μg, 12.5 μg, 25 μg, and 50 μg of OVA@MMSNs@BM-Mannose solution prepared in Example 1 to each tube, so that the concentrations of OVA@MMSNs@BM-Mannose are 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The centrifuge tube with an equal amount of ddH2O was used as the positive control group, and the centrifuge tube with an equal amount of saline was used as the negative control group. After placing the EP tube in a constant temperature water bath at 37°C for 2 hours, centrifuge at 4000 rpm for 5 minutes. 100 μL of the supernatant of each group was taken with a pipette and added dropwise into a 96-well plate. The absorbance (OD) of the supernatant of each group at 541 nm was measured with an ELISA reader. The hemolysis rate of each group was calculated using the following formula (1). If the hemolysis rate was <6%, there was no hemolysis.

[0069]

[0070] The results are as follows Figure 6 As shown in part A, compared with the erythrocytes treated with ddH2O, the saline group and the erythrocytes incubated with various concentrations of OVA@MMSNs@BM-Mann ose showed no obvious hemolysis, and even at the highest concentration of 100 μg / mL, the hemolysis rate was still maintained below 6%.

[0071] 2) The CCK-8 method was used to determine the cytotoxic effect of OVA@MMSNs@BM-Mannose nanovaccine on DC2.4 cells.

[0072] Take well-growing DC2.4 cells, digest them with trypsin and prepare them into 4×10 4 / mL cell suspension. Inoculate it into a 96-well plate at 0.1mL cell suspension / well, and add PBS buffer to the blank wells around the inoculation well for edge sealing. Place the 96-well plate in a cell culture incubator at 37°C and 5% CO2, and continue to culture overnight to allow the cells to adhere to the wall. When the cell density grows to about 80%, discard the old cell culture medium and wash with PBS buffer 2 to 3 times. Add different concentrations of OVA@MMSNs@BM-Mannose solution (so that the final concentrations are 6.25μg / mL, 12.50μg / mL, 25.00μg / mL, 50.00μg / mL, and 100.00μg / mL), add an equal amount of PBS buffer as the blank control group, and set up 5 replicates for each group. Place in a cell culture incubator and continue to culture for 24h. Discard the old culture medium, add 100μL of 10% CCK-8 serum-free culture medium to each well with a pipette, and place in the incubator for further incubation for 2h. The absorbance value (OD) of each well at 450 nm was measured and recorded using an ELISA reader. The cell survival rate was calculated according to the following formula.

[0073] Survival rate = (OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group) × 100%

[0074] The results are as follows Figure 6 As shown in Part B, when the concentration of OVA@MMSNs@BM-Mannose nanovaccine is in the range of 0-100 μg / mL, the activity of DC2.4 cells is ≥85%. Although the cell survival rate has a slight downward trend with the increase of concentration, the survival rate of DC2.4 cells at the highest concentration is still as high as 89.88%, indicating that OVA@MMSNs@BM-Mannose nanovaccine has good biocompatibility for DC2.4 cells.

[0075] Embodiment 5

[0076] The uptake effect of cells on FITC-OVA@MMSNs@BM-Mannose prepared in Example 1 was investigated, and the specific steps were as follows:

[0077] 1) Quantitative analysis of cell uptake by flow cytometry: DC2.4 cells in good growth condition were cultured at a density of about 10 5Each well was inoculated in a six-well plate and placed in a cell culture incubator for 12 hours. Subsequently, 500 μL of serum-free culture medium containing 50 μg / mL of FITC-OVA@MMSNs@BM-Mannose biomimetic nanovaccine prepared in Example 1 was added to each well. After incubation for 0 hours, 1 hour, 2 hours, and 4 hours, the cells were washed 2 to 3 times with PBS solution to remove the FIT C-OVA@MMSNs@BM-Mannose biomimetic nanovaccine attached to the cell surface. After trypsin digestion, the cells in each well were placed in a 1.5 mL centrifuge tube, centrifuged at 2000 rpm for 7 minutes, and the cell pellet was collected. 500 μL of PBS buffer was added to each centrifuge tube, and the uptake of FITC-OVA@MMSNs@BM-Mannose by DC2.4 cells was detected by flow cytometer.

[0078] The results are as follows Figure 7 As shown in the figure, as the incubation time increases, the green fluorescence intensity in the cells increases. At 1h, 2h, and 4h, the proportion of DC2.4 cells taking in the nanovaccine is 8.89%, 21.97%, and 25.86%, respectively. Among them, the increase in fluorescence intensity is not significant compared with that of incubation for 2h and 4h, indicating that the uptake of FITC-OVA@MMSNs@BM-Mannose nanovaccine by DC2.4 cells is basically saturated at 2h.

[0079] 2) Qualitative analysis of cell uptake using confocal microscopy: First, place the cell slides in a 6-well plate, then place DC2.4 cells in good growth condition at a density of about 10 5 Cells were inoculated in six-well plates at 100 μg / well and placed in a cell culture incubator for 12 hours. Washed with PBS three times, then 500 μL of serum-free medium containing 50 μg / mL FITC-OVA@MMSNs@BM-Mannose nanovaccine prepared in Example 1 was added to each well. After incubation for 0 hours, 0.5 hours, 1 hour, and 2 hours, the cells were washed with PBS buffer three times, and 500 μL of 4% paraformaldehyde solution was added to each well to fix the cells for 10 minutes. Washed with PBS buffer three times, 4',6-diamidino-2-phenylindole (DAPI) was added to stain the cell nuclei. After washing with PBS buffer three times, the cell slide was taken out and inverted on a glass slide with 20 μL of anti-fluorescence quenching sealing solution. Confocal laser scanning microscopy (CLSM) was used to observe the uptake of FIT C-OVA@MMSNs@BM-Mannose by DC2.4 cells.

[0080] The results are as follows Figure 8As shown in the figure, after DC2.4 cells were co-incubated with the nanovaccine for 0.5h, a slight green fluorescence appeared in the cytoplasm of DC2.4 cells, confirming that the nanovaccine had begun to partially enter the DC2.4 cytoplasm. As the co-incubation time increased, the green fluorescence intensity in the cytoplasm of DC2.4 cells gradually increased, indicating that DC2.4 cells continued to take up the nanovaccine. In addition, no green fluorescence of FITC-OVA appeared in the cell nucleus, confirming that the nanovaccine was mainly present in the cytoplasm and played a role in the cytoplasm.

[0081] Embodiment 6

[0082] The effects of different concentrations of the OVA@MMSNs@BM-Mannose nanovaccine prepared in Example 1 on DC2.4 cells were investigated, and the specific steps were as follows:

[0083] DC2.4 cells in good growth condition were cultured at a density of about 10 5 Each well was inoculated with 2 mL of PBS solution per well in a six-well plate and incubated in a cell culture incubator for 12 h. The old culture medium was discarded and each well was rinsed three times with 2 mL of PBS solution. A control group was set up with 1 mL of serum-free culture medium, and each well was added with 1 mL of OVA@MMSNs@BM-Mannose solution of different concentrations, so that the final concentration of manganese ions was 0.04 mM, 0.07 mM, 0.13 mM, and 0.26 mM, respectively. Three parallel samples were set up in each group. After incubation for 6 h, the old culture medium was discarded and each well was rinsed three times with 2 mL of PBS solution. Trypsin was added for digestion and centrifugation (2500 rpm, 5 min), the cell pellet was collected, and the cells were washed 3 times with PBS buffer. The cell pellet obtained in each tube was redissolved in 500 μL of PBS solution and centrifuged again to concentrate the cells at the bottom of the EP tube. The cells treated with serum-free culture medium were used as blank controls, and MRI scans were performed to obtain T1WI images and T1 map images, and the T1 signal intensity of each group was measured on the post-processing workstation.

[0084] The results are as follows Fig. 9 As shown, with the increase of Mn 2+ As the concentration increases, the T1WI signal value of DC2.4 cells increases, and the color of the pseudo-color image cell area changes from blue to red. The quantitatively measured signal value proves that DC2.4 cells have a concentration-dependent uptake of OVA@MMSNs@BM-Mannose nanovaccine, verifying its potential for application in the field of magnetic resonance imaging.

[0085] Embodiment 7

[0086] The in vitro activation of DC2.4 cells by FITC-OVA@MMSNs@BM-Mannose prepared in Example 1 was investigated, and the specific steps were as follows:

[0087] DC2.4 cells in good growth condition were cultured at a density of about 10 5 Each well was inoculated in a six-well plate and incubated in a cell culture incubator for 12 hours. The old culture medium was discarded and 2 mL of PBS solution was added to each well for three times of washing. The experiment was divided into 5 groups, and Control, OVA, MMSNs, OVA@MMSNs@BM, and OVA@MMSNs@BM-Mannose were added to different cell culture wells at a concentration of 100 μg / mL. Among them, the concentration of OVA in the OVA, OVA@MMSNs@BM, and OVA@MMSNs@BM-Mannose groups was 20 μg / mL, and Control was a DC2.4 cell group without any substance added. Three replicate wells were set for each group. After incubation for 6 hours, the old culture medium was discarded and 2 mL of PBS solution was added to each well for three times of washing. Trypsin was added for digestion and centrifugation (2500 rpm, 5 min), the cell pellet was collected, and the cells were washed 3 times with PBS buffer. Collect cells from each well in a 1.5 mL EP tube, add 2.5 μL of anti-CD11c-PE, anti-CD80-APC, and anti-CD86-FITC antibodies, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 2000 rpm for 7 minutes and collect the cell pellet. Add 500 μL of 4% paraformaldehyde solution to each EP tube, and use a flow cytometer to detect the activation of DC2.4.

[0088] The results are as follows Fig.10 As shown in the figure, the activation efficiency of DC2.4 in the Control group (0.951%) and the MMSNs group (0.1875%) was low, and the activation rates of DC2.4 cells in the OVA group, OVA@MMSNs@BM group and OVA@MMS Ns@BM-Mannose group were 2.01%, 0.715% and 5.01%, respectively. Among them, the DC2.4 activation efficiency in the OVA@MMSNs@BM-Mannose group was significantly higher than that in the OVA@MMSNs@BM group. This is mainly because the mannose on the surface of OVA@MMSNs@BM-Man nose can target and bind to the mannose receptor CD206 on the surface of DC2.4 cells, which can deliver more antigens and adjuvants into the cells, thereby activating more DC2.4 cells.

[0089] Embodiment 8

[0090] The effect of the OVA@MMSNs@BM-Mannose nanovaccine prepared in Example 1 on IFN-β secretion was investigated, and the specific steps were as follows:

[0091] DC2.4 cells in good growth condition were cultured at a density of about 10 5Each well was inoculated in a six-well plate and incubated in a cell culture incubator for 12 hours. The old culture medium was discarded and 2 mL of PBS solution was added to each well for three rinses. The experiment was divided into 6 groups, and Control, OVA, MMSNs, OVA@MMSNs, OVA@MMSNs@BM, and OVA@MMSNs@BM-Mannose were added to different cell culture wells at a concentration of 100 μg / mL. Among them, the concentration of OVA in the OVA, OVA@MMSNs, OVA@MMSNs-BM, and OVA@MMSNs-Mannose groups was 20 μg / mL, and Control was a DC2.4 cell group without any substance added. Three replicates were set up in each group. After incubation for 6 hours, the old mixed solution was taken out, centrifuged at 2000 rpm for 5 minutes, and the supernatant solution was collected. The changes in the content of IFN-β were detected using an ELISA kit.

[0092] The results are as follows Fig.11 As shown in the figure, the contents of IFN-β in the Control group, OVA group, MMSNs group, OVA@MMS Ns@BM group, and OVA@MMSNs@BM-Mannose group were 4.68pg / mL, 8.96pg / mL, 83.85pg / mL, 77.60pg / mL, 81.81pg / mL, and 97.95pg / mL, respectively. Among them, the content of IFN-β in the OVA@MMSNs@BM-Mannose group was higher than that in the OVA@MMSN@BM group, which was mainly due to the targeted delivery effect mediated by mannose.

[0093] Embodiment 9

[0094] The effects of the OVA@MMSNs@BM-Mannose nanovaccine prepared in Example 1 on blood routine and blood biochemical indicators were investigated, and the specific steps were as follows:

[0095] Twenty C57-BJ6 mice (about 20 g) were randomly divided into four groups, with five mice in each group. Each experimental group was injected with 200 μL of the OVA@MMSNs@BM-Mannos e solution (5 mg / kg) prepared in Example 1 through the tail vein, and each blank control group was injected with 200 μL of saline solution through the tail vein. Blood was collected from the blank group mice on day 0; 1 mL of blood from the experimental group mice was collected by eyeball blood sampling on days 1, 7, and 21, and sent to the laboratory of the Affiliated Hospital of Xuzhou Medical University for routine blood tests and blood biochemistry tests after processing.

[0096] The results are as follows Fig.12As shown, the results of white blood cells (WBC), red blood cells (RBC), mean hemoglobin content (MCH), mean hemoglobin concentration (MCHC), hemoglobin (HGB), hematocrit (HCT), blood urea nitrogen (UREA), total protein (TP), creatinine (CREA), alanine aminotransferase (ALT), albumin (ALB), and aspartate aminotransferase (AST) were analyzed by one-way ANOVA. Compared with the blank control group mice, all indicators were P>0.05, and there was no statistical significance. This shows that there is no significant difference in various hematological indicators of mice after injection of OVA@MMSNs@BM-Mannose, confirming that the OVA@MMSNs@BM-Mannose nanovaccine did not produce obvious toxicity to mice at the experimental dose.

[0097] Embodiment 10

[0098] The effects of the OVA@MMSNs@BM-Mannose nanovaccine prepared in Example 1 on major organs were investigated, and the specific steps were as follows:

[0099] C57-BJ6 mice were injected with 200 μL of OVA@MMSNs@BM-Mannose biomimetic nanovaccine (5 mg / kg) via the tail vein. The mice were killed on the 1st, 7th, and 21st days after injection, and the heart, liver, spleen, lung, and kidney tissues were obtained and fixed with 4% paraformaldehyde solution for 3 days. The tissues were sent to the pathology department for preparation of pathological slides, and the sections were stained with hematoxylin-eosin staining, and the pathological sections of each tissue and organ were observed using an optical microscope.

[0100] The results are as follows Fig.13 As shown in the figure, all organs tested showed no obvious organic lesions, inflammation or other abnormalities compared with the control group (day 0, before nanovaccine injection), which further confirmed the safety of OVA@MMSNs@BM-Mannose nanovaccine in mice.

[0101] Therefore, the OVA@MMSNs@BM-Mannose bionic nanovaccine prepared by the present invention has strong cell uptake ability and good storage stability; low hemolysis, even at a concentration of 100 μg / mL, the hemolysis rate can still be maintained below 6%; low in vitro cytotoxicity and high safety; no obvious toxicity to mice in vivo; the hydrated particle size is 220 nm; DC2.4 cells have a continuous uptake and concentration dependence phenomenon for the nanovaccine, which basically reaches saturation at 2 hours, and has the potential for application in the field of magnetic resonance imaging; high activation efficiency for DC2.4; and targeted delivery capability; the prepared FITC-OVA@MMSNs@BM-Mannose bionic nanovaccine can realize the visualized delivery of the nanovaccine, which is convenient for observation and research; the provided bionic nanovaccine preparation method is simple and easy to operate, and the raw materials used are safe and non-toxic or have low toxicity, and are safe for the environment and operators.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. An OVA@MMSNs@BM-Mannose bionic nanovaccine, characterized by: The OVA@MMSNs@BM-Mannose bionic nano vaccine is a nano material coated with bacterial plasma membrane.

2. The OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 1, characterized in that: The nano material is metal-doped mesoporous silica nanoparticles loaded with ovalbumin.

3. The OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 2, characterized in that: The metal-doped mesoporous silica nanoparticles are manganese-doped mesoporous silica nanoparticles.

4. A visualized FITC-OVA@MMSNs@BM-Mannose bionic nanovaccine, characterized by: Fluorescein isothiocyanate is modified on ovalbumin used in the OVA@MMS Ns@BM-Mannose bionic nanovaccine described in any one of claims 1-3.

5. A method for preparing the OVA@MMSNs@BM-Mannose bionic nanovaccine according to any one of claims 1 to 3, characterized in that: Here are the steps: S1. Preparation of mesoporous silica nanoparticles MSNs; S2, preparation of manganese-doped mesoporous silica nanoparticles MMSNs; S3, loading ovalbumin onto the prepared manganese-doped mesoporous silica nanoparticles to obtain manganese-doped mesoporous silica nanoparticles loaded with ovalbumin OVA@MMSN s; S4, preparation of bacterial plasma membrane BM; S5, coating BM with OVA@MMSNs to obtain OVA@MMSNs@BM; S6. Preparation of OVA@MMSNs@BM-Mannose bionic nanovaccine.

6. The method for preparing an OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 5, characterized in that: The preparation method of OVA@MMSNs in step S3 is: dissolving ovalbumin with ddH2O, adding MMSNs powder, ultrasonicating for 10 minutes, reacting at room temperature for 24 hours under stirring conditions, collecting the precipitate by centrifugation, and washing with ddH2O for 3 times.

7. The method for preparing an OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 6, characterized in that: The mass volume ratio of ovalbumin:ddH2O:MMSNs is 40 mg:1 mL:4 mg.

8. The method for preparing an OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 5, characterized in that: In step S5, OVA@MMSNs@BM is prepared by mechanical co-extrusion, specifically, BM and OVA@MMSNs are mixed at a mass ratio of 1:1, ultrasonicated for 20 minutes, and then passed through 400nm and 200nm polycarbonate membranes, respectively, and extruded back and forth 15-20 times.

9. The method for preparing an OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 5, characterized in that: The method for preparing the OVA@MMSNs@BM-Mannose bionic nanovaccine in step S6 is: dissolving mannose molecules in ddH2O, then adding OVA@MMSNs@BM to react, and centrifuging and washing after the reaction is completed.

10. The method for preparing an OVA@MMSNs@BM-Mannose bionic nanovaccine according to claim 9, characterized in that: The mass volume ratio of mannose: ddH2O: OVA@MMSNs@BM was 1 mg: 1 mL: 10 mg; after adding OVA@MMS Ns@BM, the mixture was reacted at 37°C for 4 h.

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