Nano-vaccine adjuvant and its application in preparing medicine for improving release of dendrobium polysaccharide

Abstract

The application belongs to the technical field of biology, and discloses a nano-vaccine adjuvant, which is a core-shell structure formed by taking a cationic polymer modified by nano calcium oxide as a core-shell, embedding dendrobium polysaccharide and a bioactive substance in the core. The cationic polymer in the nano-vaccine adjuvant is modified by nano calcium oxide, so that the hydrophilicity of the polymer is increased, and the conversion of the polymer from hydrophobic to hydrophilic enables the dendrobium polysaccharide to be quickly released from the NP.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a nano vaccine adjuvant and application thereof in preparing a drug for improving the release of dendrobium polysaccharide. Background Art

[0002] Vaccination is a safe and effective medical treatment with a high preventive rate for infectious diseases. Vaccination can induce sustained humoral and cellular immunity to prevent infectious diseases. Vaccinations primarily include inactivated vaccines, live attenuated vaccines, toxoid vaccines, and subunit vaccines. Modern vaccine preparation technologies are constantly evolving, leading to the replacement of traditional vaccines with safer recombinant proteins and protein subunits. Compared to inactivated and live attenuated vaccines, subunit vaccines have well-defined target antigens, potentially overcoming their limitations. However, their immunogenicity is poor. Therefore, the addition of appropriate adjuvants is necessary to enhance the efficacy of subunit vaccines. Aluminum, a commercial and common adjuvant, has been used in vaccines for many years. However, aluminum adjuvants have several drawbacks in clinical application, including low cellular and humoral immune responses, local and systemic side effects, and low efficacy for certain antigens. Therefore, the design and preparation of safe and effective vaccine adjuvants is crucial and necessary. Adjuvant compositions have evolved from simple natural extracts to synthetic compounds and antigen delivery systems.

[0003] Nanoparticle drug delivery systems can alter the pharmacokinetics and biodistribution of polysaccharides and enhance targeted cellular and humoral immunity, attracting significant attention as vaccine adjuvants. Furthermore, nanomaterials mimic key structural features of microorganisms sensed by the immune system, making them ideal for co-delivering antigens and / or adjuvants, eliciting signaling and immune responses, and improving drug bioavailability. Inspired by nanoparticle drug delivery, an adjuvant-based nanoparticle drug delivery system incorporates polysaccharides into the particles to protect them from degradation, thereby supporting vaccine development. Metal nanoparticles (NPs) are currently widely used drug carriers. They possess excellent biocompatibility, storage stability, ease of preparation, versatility, and minimal toxicity. They can also enable targeted, controllable, and imaging capabilities in materials with drug delivery properties. However, metal nanoparticles slow metabolism, limiting their clinical application. Therefore, coating metal NPs with a thin layer of polymer can enhance the functional properties of nanosystems (biocompatibility, bioaffinity, and biosensing). CaO2 microparticles are used as pharmaceutical excipients for oral medications and vaccines due to their ideal biocompatibility and biodegradability. Furthermore, CaO2 is pH-sensitive and reactive in acidic environments, such as lysosomes / endosomes, generating carbon dioxide, leading to rupture, antigen release, and drug delivery. However, unmodified metal nanoparticle-loaded CaO2 nanoparticles often have low solubility in water, limiting the adjuvant activity of the nanoparticles.

[0004] Cationic polymers can have different physicochemical properties and thus different functions by changing their chemical composition, molecular weight, and molecular structure. Poly(β-amino ester) (PBAE) is a class of biodegradable cationic polymers. PBAE has many ester bonds in its structure, and the material can be hydrolyzed into small molecules in the body and then excreted. Studies have shown that PBAE-coated nanoparticles can improve cellular uptake efficiency and immune response as drug delivery systems.

[0005] Dendrobium officinale, a member of the Orchidaceae family, can promote fluid secretion, enhance immunity, reduce fever, and prevent cardiovascular disease. Dendrobium officinale polysaccharides (DHPs) are composed of glucose (65.04%), mannose (14.23%), galactose (8.17%), galacturonic acid (6.41%), rhamnose (2.34%), and xylose (1.25%). Their backbone consists of 1,4-linked α-Glcp, 1,4-linked β-Glcp, and 1,4-linked β-Manp. The 1,4-linked α-Glcp and 1,4-linked β-Manp are replaced by 1,6-linked β-Galp at the C-6 position. Studies have shown that DHPs can promote the secretion of TNF-α, IL-6, and IL-4 cytokines involved in both cellular and humoral immunity in the mouse macrophage cell line RAW264.7. However, as a water-soluble polysaccharide, DHPs has disadvantages such as rapid metabolism, low biological action range, and unconcentrated action range, which limits its application in vaccine adjuvants.

[0006] Therefore, it is necessary to construct a nano-vaccine adjuvant system to increase the release rate of Dendrobium polysaccharides and exert its effect of enhancing the body's immunity. Summary of the Invention

[0007] The purpose of the present invention is to provide a nano vaccine adjuvant and its application in the preparation of a drug for improving the release of dendrobium polysaccharide. After the cationic polymer is modified with nano calcium oxide, the hydrophilicity of the polymer is increased. The conversion of the polymer from hydrophobic to hydrophilic enables the dendrobium polysaccharide to be quickly released from the NP.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides a nano vaccine adjuvant. The nano vaccine adjuvant has a core-shell structure formed by using a cationic polymer modified with nano calcium oxide as a core-shell and embedding dendrobium polysaccharide and a bioactive substance in the core.

[0010] In one technical solution, the cationic polymer is a PBAE-GB-PEG-SS-PEI polymer.

[0011] In one technical solution, the core of the nano-vaccine adjuvant uses copper nanoparticles to load dendrophanes and uses gold nano-ions to load bioactive substances.

[0012] In one technical solution, the bioactive substance is ovalbumin.

[0013] In one technical solution, the nano-vaccine adjuvant enhances immune response by regulating the MDA5-IFN-alpha axis signaling pathway.

[0014] The application also provides application of the above nano-vaccine adjuvant in preparation of a medicine for improving release of dendrophanes.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] The nano-vaccine adjuvant of the application uses PBAE-G-B-PEG-SS-PEI polymer modified by nano calcium oxide as a core-shell, and dendrophanes macromolecules and OVA are embedded in the core; the hydrophilicity of the PBAE-G-B-PEG-SS-PEI polymer is increased after being modified by nano calcium oxide; the conversion of the polymer from hydrophobic to hydrophilic enables the dendrophanes to be quickly released from the NP.

[0017] The antigen-specific IgG level in the immune serum of the group of mice immunized with the nano-vaccine adjuvant P-CaO2@Au@OVA@Cu@DHPs is obviously higher than that of the group of mice immunized with normal saline, indicating that the P-CaO2@Au@OVA@Cu@DHPs can enhance immune response as a vaccine adjuvant; in addition, the P-CaO2@Au@OVA@Cu@DHPs nanoparticles have stable positive charges and can electrostatically adsorb mRNA with negative charges; the antigen-specific antibody IgG is determined after immunization with different doses of P-CaO2@Au@OVA@Cu@DHPs / mRNA vaccine complexes, indicating that the three doses can all induce an increase in SARS-Cov-2 RBD IgG in serum, so the P-CaO2@Au@OVA@Cu@DHPs of the application can become a highly efficient vaccine carrier; in addition, the application explores the immune enhancement mechanism of the P-CaO2@Au@OVA@Cu@DHPs, and the results show that the P-CaO2@Au@OVA@Cu@DHPs enhances immune response by regulating the MDA5-IFN-alpha axis signaling pathway. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The synthesis route of PBAE-G-B-PEG-SS-PEI in the application is shown in the schematic diagram.

[0019] Figure 2 The synthesis and characterization results of P-CaO2@Au@OVA@Cu@DHPs in the application are as follows, Figure 2A is a schematic diagram of the synthesis of P-CaO2@Au@OVA@Cu@DHPs; Figure 2 B and Figure 2 C are the SEM images and EM images of Cu@DHPs, Au@OVA@Cu@DHPs, CaO2@Au@OVA@Cu@DHPs and P-CaO2@Au@OVA@Cu@DHPs nanoparticles, respectively; Figure 2 D is the elemental analysis of P-CaO2@Au@OVA@Cu@DHPs nanoparticles; Figure 2 E particle size distribution; Figure 2 F is the PDI of P-CaO2@Au@OVA@Cu@DHPs nanoparticles in aqueous solution analyzed by DLS; Figure 2 G is the Zeta potential of P-CaO2@Au@OVA@Cu@DHPs nanoparticles in different solvents; Figure 2 H and Figure 2 I represents the DHPs-EE (%) and OVA-EE (%) of P-CaO2@Au@OVA@Cu@DHPs nanoparticles, respectively. The differences between the different superscript bars are significant (p < 0.05). Data are expressed as mean ± SEM, n = 3.

[0020] Figure 3 The storage stability of P-CaO2@Au@OVA@Cu@DHPs at 4°C is shown in Figure 2. Figure 3 A is the SEM images of P-CaO2@Au@OVA@Cu@DHPs at 0d, 7d, 14d, and 28d; Figure 3 B is the measurement of particle size, PDI, zeta potential, and zeta potential of nanoparticle suspension within 28 days; Figure 3 C and Figure 3 D are the OVA release rate (%) and DHPs release rate (%) of P-CaO2@Au@OVA@Cu@DHPs nanoparticle suspension measured over 24 days.

[0021] Figure 4 The cytotoxicity and macrophage antigen uptake of P-CaO2@Au@OVA@Cu@DHPs of the present invention are shown in FIG. Figure 4 A is the viability of macrophages incubated with DHPs and P-CaO2@Au@OVA@Cu@DHPs for 48 h; Figure 4 B is the CLSM image of macrophages incubated with OVA-FITC, PBAE-G-PEG-SS and P-CaO2@Au@OVA@Cu@DHPs.

[0022] Figure 5The effect of P-CaO2@Au@OVA@Cu@DHPs on macrophage activation and the transcriptome pathway activated by P-CaO2@Au@OVA@Cu@DHPs is shown in the figure. Figure 5 A and Figure 5 B shows the expression of CD86+ and MHCII+ on macrophages; Figure 5 C and Figure 5 D is the levels of cytokines TNF-α and IL-6 secreted by macrophages.

[0023] Figure 6 The potential toxicity of P-CaO2@Au@OVA@Cu@DHPs in vivo was investigated. Figure 6 A is H&E staining of the heart, lung, liver, spleen, and kidney of the inoculated mouse on day 35 after the second immunization, scale bar: 40 μm; Figure 6 B is the levels of serum biochemical indicators (ALT, AST, ALP, LDH, BUN) of vaccinated mice on the 35th day after the second immunization. The results are expressed as mean ± SEM, n = 3, n = 3. There was no significant difference among the groups.

[0024] Figure 7 The CLSM diagram of the P-CaO2@Au@OVA@Cu@DHPs / mRNA vaccine complex of the present invention and the induced immune response are shown in FIG. Figure 7 A is the CLSM image of the vaccine complex; Figure 7 B is a schematic diagram of the immune response experimental process; Figure 7 C shows the titer of RBD-specific IgG at the indicated time points. Histograms with different letters in a-f bars show significant differences (P < 0.05). Data are expressed as mean ± SEM, n = 5.

[0025] Figure 8 The present invention uses ELISA to detect the levels of Th1 and Th2 cytokines in the serum of immune mice, wherein Figure 8 A-8B are Th1 cytokines IFN-γ and TNF-α, respectively; Figure 8 C-8D are the levels of Th2 cytokines IL-4 and IL-6, respectively. Bars with different letters have significant differences (P<0.05) (mean ± SEM, n=4).

[0026] Figure 9 The present invention P-CaO2@Au@OVA@Cu@DHPs / mRNA stimulates cell response through the MDA5-IFN-α axis signaling pathway, wherein Figure 9A and 9B are immunoblot analyses of MDA5-IFN-α in macrophages treated with P-CaO2@Au@OVA@Cu@DHPs / mRNA. Bars with different letters indicate significant differences (P < 0.05). (Mean ± SEM, n = 4). DETAILED DESCRIPTION

[0027] The following examples are used to illustrate the present invention but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified.

[0028] Example 1

[0029] 1.1 Materials

[0030] Dendrobium officinale polysaccharides (DHPs, 95%, CY200618) were obtained from Professor Ma Bingji of Henan Agricultural University. Anhydrous calcium chloride (CaCl2), sodium hydroxide (NaOH), and hydrogen peroxide (H2O2, 30%) were obtained from Beijing Chemical Plant (Beijing, China). Copper chloride pentahydrate (CuSO4·5H2O) and chloroauric acid were purchased from Guangdong Xilong Chemical Reagent Co., Ltd. (Guangdong). Succinylidene succinate-polyethylene glycol-COOH (SS-PEG-COOH, NOR-N-0027) was purchased from Xi'an Ruixi Biotechnology Co., Ltd. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), RPMI 1640, fetal bovine serum (FBS), 0.25% trypsin-EDTA, JC-1 probe, and Annexin V FITC / PI kit were purchased from Thermo Fisher Scientific (Shanghai, China). Rhod-2 AM and MitoTracker Red were obtained from Yeasen (Shanghai, China). 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Bestbio (Shanghai, China). Alizarin Red was obtained from Yunaye (Shanghai, China). Mouse TNF-α enzyme-linked immunosorbent assay kit and mouse IFN-γ enzyme-linked immunosorbent assay kit were obtained from Tianjin Anaerobic Biotechnology Co., Ltd., China. Ovalbumin (OVA) was obtained from Sigma-Aldrich (MO, USA). All other reagents were of analytical grade.

[0031] 1.2 FT-IR, NMR Spectra and Molecular Morphology of DHPs

[0032] 1D and 2D NMR spectra were acquired on a Bruker ARX500 spectrometer (Bruker, Rheinstein, Germany) at 500 MHz and 125 MHz, respectively. 2 mg of Dendrobium officinale polysaccharide was completely dissolved in 0.5 mL of D2O at 25°C. Spectra were observed at 25°C using 60,000 scans. Data were processed using standard Bruker Topspin-NMR software.

[0033] The DHPs sample was added to KBr and pressed into 1 mm pellets for FT-IR measurement. The spectrum of ASP was recorded in the range of 400-4000 cm using FT-IR spectrometer (Nicolet NEXUS 670). -1 within the frequency range.

[0034] The molecular morphology of DHPs was observed using a FEI Quanta 250FEG scanning electron microscope (SEM). The samples were sputtered with Pt using a Cressington 108auto sputter coater, and images were observed at 800x and 1000x magnification under high vacuum at 3.0 kV.

[0035] 1.3 Synthesis of Au@OVA@Cu@DHPs

[0036] A 5 mL solution of CuSO₄·5H₂O (10 mg / mL) was mixed with a 10 mL solution of DHPs at room temperature and stirred for 5 minutes. A 1 M solution of NaOH was then added to adjust the pH to 10. The solution was then stirred for 24 hours. The product (Cu@DHPs) was collected by centrifugation, washed three times with deionized water, and finally freeze-dried.

[0037] 10 ml of a 10 mg / mL Cu@DHPs aqueous solution and 10 ml of an OVA aqueous solution were mixed and stirred at room temperature for 5 minutes. A sodium citrate aqueous solution was added to the solution and stirred at 150°C for 30 minutes. A sodium chloride aqueous solution was added to the solution and stirred for 15 minutes. The product (Au@OVA@Cu@DHPs) was collected by centrifugation, washed three times with deionized water, and finally freeze-dried.

[0038] 1.4 Synthesis of CaO2@Au@OVA@Cu@DHPs

[0039] 5 mL of sodium acetate solution (5 mg / mL), 2.5 mL of CaCl₂ solution (0.1 M), and 2.5 mL of Au@OVA@Cu@DHPs solution were mixed and stirred at room temperature for 5 minutes. Then, 5 mL of NaOH solution (0.25 M) was added and stirred for 5 minutes. 200 μL of 30% H₂O₂ was added dropwise and stirred at room temperature for 30 minutes. The product (CaO₂@Au@OVA@Cu@DHPs) was collected by centrifugation, rinsed three times with deionized water, and finally collected by freeze-drying.

[0040] Synthesis of 1.5PBAE-GB-PEG-SS-PEI

[0041] Synthesize PBAE-GB-PEG-SS-PEI conjugates as Figure 1 The process can be divided into three steps: 1) the amino group of PBAE-G and DSPE-PEG 2000 -NHS undergoes an amidation reaction to obtain PBAE-GB; 2) the amino group of PBAE-GB undergoes an amidation reaction with the carboxyl group of SS-PEG-COOH to obtain PBAE-GB-PEG-SS; 3) the amino group of PBAE-GB-PEG-SS undergoes an amidation reaction with the carboxyl group of polyetherimide (Cy5.5-PEI) (1.8KD) with a fluorescent dye, and polyetherimide is introduced to increase the hydrophobicity of PBAE-GB-PEG-SS to obtain a PBAE-GB-PEG-SS-PEI conjugate.

[0042] Synthesis of 1.6P-CaO2@Au@OVA@Cu@DHPs

[0043] 20 mg of CaO2@Au@OVA@Cu@DHPs was added to 200 mg of PBAE-GB-PEG-SS-PEI conjugate in PBS (pH = 8.0). The mixture was stirred for 8 hours and then dialyzed against PBS (pH = 7.4) (molecular weight cutoff, MWCO 4500 Da) to remove the organic solvent. The product (P-CaO2@Au@OVA@Cu@DHPs) was collected by centrifugation, rinsed three times with deionized water, and finally collected by freeze-drying.

[0044] 1.7 Characterization of Nanoparticles

[0045] The particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS instrument (Hydro2000Mu, Malvern Instruments, UK). The morphology of the nanoparticles was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

[0046] An improved microcolumn centrifugation method was used.

[0047] The calculation formula for loading efficiency is as follows:

[0048] DHPs loading efficiency = (1-Q1 / Q2) × 100%,

[0049] Where: Q1 is the number of idle DHPs, Q2 is the total number of DHPs in the P-CaO2@Au@OVA@Cu@DHPs pause.

[0050] OVA loading efficiency = (1-Q3 / Q4) × 100%,

[0051] Where: Q3 is the amount of free OVA, and Q4 is the total amount of OVA in the P-CaO2@Au@OVA@Cu@DHPs suspension.

[0052] 1.8 Determination of the release capacity of DHPs and OVA from P-CaO2@Au@OVA@Cu@DHPs

[0053] 1 mg of P-CaO2@Au@OVA@Cu@DHPs was dispersed in 2 mL of water at different time points and stirred for 12 hours. The supernatant was collected by centrifugation, and the concentrations of DHPs and OVA in serum were determined using the phenol-sulfuric acid method.

[0054] 1.9 Stability of Nanoparticles

[0055] The nanoparticle suspension was stored at 4°C to investigate its stability. Particle size, PDI, and zeta potential were evaluated at 7, 14, 21, and 28 days. DHPs-EE (%) and OVA-EE (%) of the nanoparticles were measured at the same time intervals over 28 days. All experiments were performed in triplicate.

[0056] 1.10 Isolation of mouse peritoneal macrophages

[0057] Mouse peritoneal macrophages were generated from ICR mice (6 weeks old). 1 mL of 6% starch broth medium was injected into the peritoneal cavity of the mice for 3 consecutive days. Macrophages were collected and their peritoneal cavity was irrigated twice with PBS. The separated cells were collected, centrifuged at 1500 rpm for 8 minutes, and suspended in complete culture medium at a cell density of 2×105 cells / mL. Subsequently, the cell suspension was cultured in a humidified atmosphere (37°C, 5% CO2) for 4 hours, then washed with PBS to remove non-adherent cells, and fresh complete culture medium was added. The obtained macrophages were used for the next experiment.

[0058] 1.11 Macrophage viability study

[0059] The MTT method was used to evaluate the cell viability of peritoneal macrophages. The cells were seeded in 96-well plates, 100 μL per well, and then 100 μL of P-CaO2@Au@OVA@Cu@DHPs nanoparticles and free DHPs were added at different concentrations (0-500 μg / mL). The cell group and complete culture medium served as cell controls. After culturing in a humidified incubator (37°C, 5% CO2) for 48 hours, 30 μL of MTT (5 mg / mL) was added. After incubation for 4 hours, the culture medium was removed and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader (Thermo, USA). The cell viability was calculated by the absorbance ratio of the experimental group to the control group. All experiments were repeated four times.

[0060] 1.12 Flow cytometry detection of MHCII and CD86

[0061] Flow cytometry was used to detect the expression of surface markers MHCII and CD86. Macrophages were incubated with P-CaO2@Au@OVA@Cu@DHPs and free DHPs at a concentration of 30 μg / mL for 48 hours. A trypsin-EDTA solution (containing phenol red) was then added to the plate, washed by centrifugation at 4000 rpm for 5 minutes, and labeled with anti-MHCII-FITC and anti-CD86-PE for 30 minutes in the dark. The plates were washed three times with PBS, and the expression of MHCII and CD86 on macrophages was determined by flow cytometry.

[0062] 1.13 Macrophages secrete cytokines

[0063] Macrophages were incubated with P-CaO2@Au@OVA@Cu@DHPs and free DHPs at a concentration of 30 μg / mL for 48 hours. After incubation, the cell culture supernatants were collected and the levels of cytokines TNF-α and IL-1β were measured using ELISA kits, RT-PCR, and Western blot. The optical density (OD) was measured at 450 nm using a microplate reader. All experiments were performed in triplicate.

[0064] 1.14 Macrophage Uptake Capacity

[0065] A mixture of P-CaO2@Au@OVA@Cu@DHPs, OVA-FITC (green), and PBAG-G-PEG-SS (red) was incubated in a 24-well plate in a humidified incubator (37°C, 5% CO2) for 12 hours. Following incubation, the cells were washed multiple times with PBS and stained with Hoechst 33342 for 10 minutes. Excess dye was removed by washing the cells multiple times with PBS. Finally, fluorescence images were recorded using an inverted microscope.

[0066] 1.15 Animals and vaccination

[0067] 1.15.1 Preparation of SARS-CoV-2 RBD mRNA Vaccine

[0068] SARS-CoV-2 RBD mRNA was prepared as described in (Ren et al., 2021). P-CaO2@Au@OVA@Cu@DHPs / mRNA complexes were freshly prepared by mixing a certain amount of mRNA stock solution with varying amounts of P-CaO2@Au@OVA@Cu@DHPs in sterile distilled water. After gentle vortexing, the complexes were incubated at room temperature for 30 minutes to allow for particle formation.

[0069] 1.15.2 Animal Immunization

[0070] Female BALB / c mice aged 6 to 8 weeks were randomly divided into 6 groups (5 mice per group) and injected intramuscularly three times with the P-CaO2@Au@OVA@Cu@DHPs / mRNA composite vaccine (5μg, 10μg, 30μg mRNA / mouse, N / P=32), once every 14 days. The negative control group was given equal amounts of saline, 30μg mRNA and PVES at the same time. Blood was collected from the orbital vein 14 days and 28 days after the first immunization. The collected blood was centrifuged at 4000rpm to separate the serum (30 minutes, 4°C). The serum was stored at -20°C for subsequent SARS-CoV-2RBD-specific IgG.

[0071] 1.16 Serum Antibody Evaluation

[0072] ELISA kit for detecting SARS-CoV-2 RBD-specific IgG antibodies. The ELISA kit was used to detect SARS-CoV-2 RBD-specific IgG titers according to the manufacturer's instructions. Briefly, 10-fold diluted serum starting from 1:100 was added to a blocked 96-well plate coated with recombinant SARS-CoV-2 RBD protein (100 μL / well) and incubated at room temperature for 2 hours. After washing five times with wash buffer, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was added to the culture dish and incubated at room temperature for 1 hour. The plate was then washed five times with wash buffer, and the color developer solution was added and incubated at room temperature for 20 minutes. The absorbance at 450 nm was read using a microplate reader. The endpoint titer was defined according to the manufacturer's instructions.

[0073] 1.17 Intracellular cytokine staining assay

[0074] Intracellular cytokine staining was performed to characterize antigen-specific CD4+ and CD8+ immune responses. Briefly, spleens were harvested from immunized mice 4 weeks after immunization, and splenocytes were isolated. Mouse splenocytes were plated in 12-well plates (1 × 106 cells / well) and stimulated with peptide pools (2 μg / mL of individual peptides) for 2 h. Golgiplug (BD Biosciences) was then added at a final concentration of 1 μL / mL. After incubation for 4 h, cells were harvested and stained with anti-CD4 and anti-CD8α surface markers (Biolegend). Subsequently, cells were fixed and permeabilized in permeabilization buffer (BD Biosciences) and stained with anti-IFN-γ and anti-IL-4 (Biolegend). Flow cytometric analysis was performed using a BD FACSAria II flow cytometer, and data were analyzed using FlowJo 10.0.

[0075] 1.18 Determination of cytokines in serum

[0076] The concentrations of IFN-γ, TNF-α, IL-4, and IL-6 cytokines in serum were measured on days 21 and 28 after primary vaccination using a Ready-to-use Sandwich ELISA kit according to the manufacturer's instructions. Optical density (OD) was measured at 450 nm, and all experiments were repeated four times.

[0077] 1.19 Statistical Analysis

[0078] All values ​​are expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed using Duncan's multiple-range test and LSD. A probability value (p) of less than 0.05 was considered statistically significant.

[0079] 2 Results and Discussion

[0080] 2.1Synthesis and characterization of P-CaO2@Au@OVA@Cu@DHPs.

[0081] like Figure 2 As shown in Figure A, the nanoparticles (NPs) of the present invention have a core-shell structure, with the core capable of encapsulating dendrobium polysaccharide macromolecules and OVA. Modification of the PBAE-GB-PEG-SS-PEI polymer with nano-calcium oxide further increases its hydrophilicity. This transformation of the polymer from hydrophobic to hydrophilic allows for the rapid release of the dendrobium polysaccharide from the NPs.

[0082] from Figure 2 B and Figure 2 As can be seen from Figure C, the well-dispersed P-CaO2@Au@OVA@Cu@DHPs presents a spherical morphology with a diameter of about 150nm. Scanning electron microscopy (SEM) results show that the core-shell copper-gold coated DHPs and OVA are spherical with slightly uneven surfaces, while the P-CaO2@Au@OVA@Cu@DHPs nanoparticles are also nearly spherical with slightly uneven surfaces. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) clearly show the flaky morphology of P-CaO2@Au@OVA@Cu@DHPs. Figure 2 As can be seen in D, the elemental mapping results of single P-CaO2@Au@OVA@Cu@DHPs show that dense C (red) and O (lake blue) are distributed around the Au (purple) core, confirming that P-CaO2@Au@OVA@Cu@DHPs are uniformly distributed.

[0083] like Figure 2 As shown in E, the average particle size of P-CaO2@Au@OVA@Cu@DHPs is slightly larger than that of CaO2@Au@OVA@Cu@DHPs. The average particle size of P-CaO2@Au@OVA@Cu@DHPs is 157.2±2.21nm. The PDI is lower than 0.3, indicating that the size distribution is narrow ( Figure 2 F).

[0084] The stability of P-CaO2@Au@OVA@Cu@DHPs nanoparticles was studied by measuring the ζ-potential (different dissolution rates), e.g. Figure 2 G. The ζ-potentials of P-CaO2@Au@OVA@Cu@DHPs nanoparticles were positively charged in water (11.90±0.71 mV), 10% FBS (12.30±1.45 mV), and PBS (11.73±1.11 mV). Furthermore, there was no difference in ζ-potential between day 1 and day 35 (p>0.05).

[0085] like Figure 2H, the DHPs encapsulation efficiency of Cu@DHPs, Au@OVA@Cu@DHPs, CaO2@Au@OVA@Cu@DHPs and P-CaO2@Au@OVA@Cu@DHPs nanoparticles were 55.7 ± 1.31%, 54.17 ± 2.60%, 50.53 ± 1.59%, 47.8 ± 1.11%, respectively. As shown in Figure 2 I, the OVA encapsulation efficiency of Au@OVA@Cu@DHPs, CaO2@Au@OVA@Cu@DHPs and P-CaO2@Au@OVA@Cu@DHPs nanoparticles were 62.83 ± 0.81%, 56.03 ± 2.09%, 53.73 ± 0.52%, respectively. These results indicated that P-CaO2@Au@OVA@Cu@DHPs were successfully prepared.

[0086] 2.2 Stability of nanoparticles

[0087] To evaluate the stability of P-CaO2@Au@OVA@Cu@DHPs nanoparticles suspension, the morphology of nanoparticles was measured by transmission electron microscopy (TEM) at 4 °C for 4 weeks. As shown in Figure 3 A, the morphology of P-CaO2@Au@OVA@Cu@DHPs nanoparticles changed little. As shown in Figure 3 B, the average size of P-CaO2@Au@OVA@Cu@DHPs nanoparticles varied from 172.5 nm to 186.2 nm over a period of 28 days. The PDI of nanoparticles had no significant change and deviation within 28 days, both of which were less than 0.3, indicating that the particle size distribution of nanoparticles was uniform within 28 days. The zeta potential of nanoparticles also had no change within 28 days.

[0088] 2.3 Ability of P-CaO2@Au@OVA@Cu@DHPs nanoparticles to release DHPs and OVA

[0089] As shown in Figure 3As shown in FIG. C, the P-CaO2@Au@OVA@Cu@DHPs nanoparticles encapsulated OVA released slowly in the cell mimicking environment solution, and the cumulative release amount was about 5.73 ± 0.06% at day 1. However, in the cell mimicking environment, the P-CaO2@Au@OVA@Cu@DHPs nanoparticles released OVA rapidly from day 1 to day 16, and the cumulative release amount was about 42.97 ± 0.20%. From day 16 to day 36, the release of OVA accelerated again, and the cumulative release amount was about 47.15 ± 0.56%. This may be due to the degradation of PBAE-G-B-PEG-SS-PEI conjugate and the damage of CaO2 emulsion structure, resulting in the accelerated release of OVA from day 16 to day 36. The cumulative release results show that the P-CaO2@Au@OVA@Cu@DHPs nanoparticles have good controlled release effect. At the same time, the in vitro release of DHPs from P-CaO2@Au@OVA@Cu@DHPs nanoparticles is shown in FIG. D. Figure 3 As shown in FIG. D, DHPs released slowly at the first day, and then released rapidly. The cumulative release amount of DHPs was about 57.25 ± 0.12% at day 36.

[0090] 2.4 Cytotoxicity of P-CaO2@Au@OVA@Cu@DHPs nanoparticles and antigen uptake by macrophages

[0091] The cytotoxicity of DHPs and P-CaO2@Au@OVA@Cu@DHPs particles was studied by MTT method. Macrophages were exposed to different concentrations of GO and GO-LNT for 48 hours. DHPs and P-CaO2@Au@OVA@Cu@DHPs were non-toxic to macrophages at 30 μg / mL (FIG. A). Therefore, the concentration of 30 μg / mL was selected for subsequent experiments. Figure 4

[0092] The ability of macrophages to effectively uptake antigens is a prerequisite for antigen presentation and stimulation of immune response. Macrophages and neutrophils play an important role in both innate immunity and adaptive immunity, and constitute the first line of defense against pathogen invasion. To study the uptake of antigens by macrophages, laser confocal microscopy was used to observe the uptake of OVA-FITC, PBAE-G-PEG-SS and P-CaO2@Au@OVA@Cu@DHPs. Figure 4 B shows that the amount of OVA-FITC internalization is higher compared to OVA-FITC alone. The results show that P-CaO2@Au@OVA@Cu@DHPs induce macrophages to effectively uptake antigens.

[0093] 2.5 Expression level of surface molecules of macrophages and secretion of cytokines

[0094] ​Macrophages are activated after phagocytosis and present antigens to T lymphocytes, thereby activating adaptive immune responses. In addition, macrophages also play a key role in humoral and cellular immunity. After cellular antigen uptake, macrophages highly express co-stimulatory molecules. CD86 + and MHCII + The high expression of is related to the presentation of exogenous antigens to T cells and the promotion of T cell activation. To detect the effect of P-CaO2@Au@OVA@Cu@DHPs on macrophage activation, flow cytometry was used to detect the co-stimulatory molecule MHCII + and CD86 + Expression. Figure 5 As shown in A and 5B, compared with the control group, MHCII + and CD86 + The expression of CD86 in the P-CaO2@Au@OVA@Cu@DHPs group was significantly upregulated (p<0.05), and there was no difference compared with the LPS group (p>0.05). + The expression was significantly higher than that in other groups (p<0.05).

[0095] Immune cells, especially macrophages, secrete cytokines that play a key role in the initiation and regulation of immune responses, which are necessary for the clearance of infected cells and pathogens. Macrophages were stimulated in vitro with P-CaO2@Au@OVA@Cu@DHPs, and the levels of IL-6 and TNF-α in the supernatant were detected by ELISA. Figure 5 As shown in Figures C and 5D, the secretions of IL-1β and TNF-α induced by P-CaO2@Au@OVA@Cu@DHPs were higher than those in the other groups (P<0.05 and P<0.01).

[0096] 2.6 Potential toxicity of P-CaO2@Au@OVA@Cu@DHPs in vivo

[0097] To investigate the safety of P-CaO2@Au@OVA@Cu@DHPs as a vaccine adjuvant, the toxicity of GO-LNT / OVA on major organs was evaluated. Heart, lung, liver, spleen, and kidney tissues were obtained and stained with H&E. Compared with the PBS group, the tissue structure of the experimental group was normal, with no obvious inflammation and damage ( Figure 6B). In order to further explore the potential toxicity of GO-LNT / OVA in vivo, the present invention conducted serum biochemical tests on mice. Liver function indicators (ALT, AST, ALP), myocardial function indicators (LDH), and renal function indicators (BUN) were measured. The levels of ALT, AST, and ALP in serum are important indicators for detecting liver function. A significant increase in the levels of AST, ALT, and ALP represents liver damage. Because the ability to excrete urea nitrogen in the blood decreases, the kidneys are damaged, resulting in an increase in the BUN level of the kidneys. Lactate dehydrogenase is a glycolytic enzyme, and an increase in its activity is usually observed in diseases that affect the myocardium or liver. There was no significant change in ALT, AST, ALP, LDH, and BUN in the P-CaO2@Au@OVA@Cu@DHPs group, and they were still within the normal range ( Figure 6 A). These results indicate that P-CaO2@Au@OVA@Cu@DHPs is safe as a vaccine adjuvant.

[0098] 2.7 Immune Response Induced by P-CaO2@Au@OVA@Cu@DHPs Vaccine Complex

[0099] like Figure 7 As shown in Figure A, the P-CaO2@Au@OVA@Cu@DHPs vaccine complex was observed by CLSM. SARS-CoV-2 RBD mRNA exhibited green fluorescence in the vaccine complex. In addition, red fluorescence of P-CaO2@Au@OVA@Cu@DHPs was observed in the vaccine complex.

[0100] To further verify whether P-CaO2@Au@OVA@Cu@DHPs can be used as an adjuvant for mRNA vaccines and induce immune responses in vivo, five groups of mice (n=5) were immunized with different doses of P-CaO2@Au@OVA@Cu@DHPs / mRNA vaccine complex (5μg, 10μg, and 30μg / mouse). The mice were immunized on day 0, boosted on day 14, and sacrificed on days 21, 28, 35, and 42. Serum samples were collected for antibody and cytokine analysis ( Figure 7 B). Spleens were harvested on day 28 for histopathological analysis. Mice injected with PBS served as the control group. ELISA was used to measure the titer of RBD-specific antibodies to assess humoral immune responses ( Figure 7 C). The results showed that three doses of P-CaO2@Au@OVA@Cu@DHPs / mRNA complexes produced significant antibody titers 21 days after the first immunization. Antibody levels increased rapidly after booster immunizations. The titers of RBD-specific antibodies in mice immunized with the high dose were significantly higher than those in mice immunized with the low dose. The average endpoint titer in the 30μg group rose to >10 after the third immunization. 5Compared with the 10μg group and the 5μg group, the levels of IgG1, IgG2a and IgG2b antibodies were increased by 1.5 times and 5.2 times, respectively. Therefore, we chose the 30μg dose to immunize mice in the following study. Figure 7 C. The results are consistent with the hypothesis well.

[0101] 2.8 Serum cytokine levels

[0102] On days 21, 28, and 35 after the first vaccination, the concentrations of cytokines in serum were determined by ELISA. Compared with the other groups, the levels of Th1 cytokines (IFN-γ and TNF-α) induced by P-CaO2@Au@OVA@Cu@DHPs / mRNA were significantly increased (P<0.05) ( Figure 8 A and Figure 8 B). TNF-α plays a key role in the cellular immune process and can promote Th1 response. The change trend of IL-4 and IL-6 secretion P-CaO2@Au@OVA@Cu@DHPs / mRNA was similar to that of IFN-γ and TNF-α, and was significantly higher than that of other groups (P<0.05) ( Figure 8 C. Figure 8 D). These results indicate that P-CaO2@Au@OVA@Cu@DHPs / mRNA can induce the secretion of Th1 and Th2 cytokines, which is consistent with the above antibody response results.

[0103] 2.9P-CaO2@Au@OVA@Cu@DHPs / mRNA stimulates cellular responses through the MDA5-IFN-α axis signaling pathway

[0104] The SARS-CoV-2 spike protein binds to and stimulates TLR2 and TLR4 signaling. Furthermore, the immune response to seasonal influenza and other unadjuvanted vaccines is controlled by the microbiome through the TLR5 pathway. The BNT162b2 mRNA vaccine, co-developed by Pfizer and BioNTech, stimulates CD8+ T cell responses through the MDA5-IFN-α axis.

[0105] The present invention stimulates CD8 through P-CaO2@Au@OVA@Cu@DHPs / mRNA + T cell detection and MDA5-IFN-α axis signaling pathway related genes were used to explore the role and mechanism of P-CaO2@Au@OVA@Cu@DHPs / mRNA. Figure 9As shown in A and 9B, P-CaO2@Au@OVA@Cu@DHPs / mRNA significantly increased the protein expression levels of MDA5, cGAS, STING, and IFN-γ in both the LPS and control groups. Furthermore, P-CaO2@Au@OVA@Cu exhibited similar effects to P-CaO2@Au@OVA@Cu@DHPs, with mRNA increasing the protein expression levels of MDA5, cGAS, STING, and IFN-γ, similar to those in the LPS and control groups. These results suggest that P-CaO2@Au@OVA@Cu@DHPs / mRNA stimulates cellular responses through the MDA5-IFN-α axis signaling pathway.

[0106] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A nano vaccine adjuvant, characterized in that The nano vaccine adjuvant is a cationic polymer modified with nano calcium oxide as the core-shell, with copper nanoparticles loading dendrobium polysaccharide in the core and gold nano ions loading bioactive substances. The cationic polymer is a PBAE-GB-PEG-SS-PEI polymer; the bioactive substance is ovalbumin; the nano vaccine adjuvant enhances the immune response by regulating the MDA5-IFN-α axis signaling pathway.

2. Use of the nano vaccine adjuvant according to claim 1 in the preparation of a drug for improving the release of dendrobium polysaccharides.

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