CN115920027A - Nano vaccine adjuvant and application thereof in preparing medicine for improving release force of dendrobium polysaccharide - Google Patents
Nano vaccine adjuvant and application thereof in preparing medicine for improving release force of dendrobium polysaccharide Download PDFInfo
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Abstract
The invention 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 and embedding dendrobium polysaccharide and bioactive substances 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 dendrobe polysaccharide can be quickly released from NP due to the conversion of the polymer from hydrophobicity to hydrophilicity.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a nano vaccine adjuvant and application thereof in preparation of a drug for improving release force of dendrobe polysaccharide.
Background
Vaccination is a safe and effective medical treatment, and has a high rate of prevention of infectious diseases. Vaccination can induce a sustained humoral and cellular immunity to prevent infectious diseases. The vaccination mainly comprises inactivated vaccine, attenuated live vaccine, toxoid vaccine, subunit vaccine and the like. Modern vaccine preparation techniques are constantly being updated, resulting in safer recombinant proteins and protein subunits replacing traditional vaccines. Compared with inactivated vaccines and attenuated live vaccines, subunit vaccines have specific target antigens, can make up for the limitations of inactivated vaccines or attenuated live vaccines, but have poor immunogenicity. Therefore, the efficacy of subunit vaccines needs to be improved by the addition of appropriate adjuvants. Aluminum has been used in vaccines for many years as a commercial and conventional adjuvant. However, aluminum adjuvants have several drawbacks in clinical applications, including low cellular and humoral mediated immune responses, local and systemic side effects, and low efficiency of certain antigens. Therefore, it is very important and necessary to design and prepare a safe and efficient vaccine adjuvant. Adjuvant components have evolved from simple natural extracts to artificially synthesized compounds and antigen delivery systems.
The nano drug delivery system can change the pharmacokinetics and biodistribution of polysaccharide, and enhance the target cell and humoral immunity, and has attracted wide attention in the aspect of vaccine adjuvant. In addition, the nanomaterials mimic the key structural features of the microbes perceived by the immune system, are well suited to co-deliver antigens and/or adjuvants, elicit signaling and immune responses, and enhance the bioavailability of the drug. Inspired by nano-drug delivery, an adjuvant-based nano-drug delivery system supports vaccine development by adding polysaccharides to particles to protect them from polysaccharide degradation. Metal Nanoparticles (NPs) are currently widely used drug carriers. Has the characteristics of good biocompatibility, good storage stability, convenient preparation, strong multifunctionality, small toxic and side effects and the like. It can also make some materials with drug administration property obtain targeting, controllability and imaging function. However, metal nanoparticles slow metabolism, limiting their clinical use. Therefore, coating a thin polymer layer on the surface of the metal NPs can enhance the functional characteristics (biocompatibility, bioaffinity and biosensing) of the nano system. CaO (CaO) 2 Microparticles are used as pharmaceutical excipients for oral drugs and vaccines because of their ideal biocompatibility and biodegradability. In addition, caO 2 Sensitive to pH, reactive in acidic environments (e.g. lysosomes/endosomes) and thereby generating carbon dioxide, leading to rupture, antigen release and drug delivery. However, unmodified metal nanoparticle-supported CaO 2 The solubility of nanoparticles in water tends to be low, limiting the adjuvant activity of nanoparticles.
Cationic polymers can have different physicochemical properties by changing their chemical composition, molecular mass, and molecular structure, and thus have different functions. Poly (β -amino esters) (PBAE) are a class of biodegradable cationic polymers. PBAE have many ester linkages in their structure, which can be hydrolyzed in vivo into small molecules and then excreted in vitro. Research shows that PBAE coated nanoparticles can be used as a drug delivery system to improve cellular uptake efficiency and improve immune response.
The plant of Dendrobium is Orchidaceae, and has effects of promoting secretion of body fluid, enhancing immunity, relieving fever, and preventing cardiovascular disease. Dendrobii polysaccharides (DHPs) consist of glucose (65.04%), mannose (14.23%), galactose (8.17%), galacturonic acid (6.41%), rhamnose (2.34%) and xylose (1.25%). The backbone consists of 1, 4-linked α -Glcp, 1, 4-linked β -Glcp and 1, 4-linked β -Manp, wherein the 1, 4-linked α -Glcp and 1, 4-linked β -Manp are 1, 6-linked β -Galp at the C-6 site. Research shows that DHPs can promote the secretion of TNF-alpha, IL-6 and IL-4 cytokines involved in cell and humoral immunity in a mouse macrophage system RAW 264.7. However, DHPs as a water-soluble polysaccharide has the disadvantages of fast metabolism, low biological action range, no centralized action range and the like, and the application of DHPs in vaccine adjuvants is limited.
Therefore, a nano vaccine adjuvant system is needed to be constructed to improve the release rate of the dendrobium polysaccharide and exert the effect of enhancing the immunity of the organism.
Disclosure of Invention
The invention aims to provide a nano vaccine adjuvant and application thereof in preparing a drug for improving the release force of dendrobium polysaccharide.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides 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 and embedding dendrobium polysaccharide and bioactive substances in the core.
In one embodiment, the cationic polymer is a PBAE-G-B-PEG-SS-PEI polymer.
In one technical scheme, the core of the nano vaccine adjuvant is loaded with dendrobium polysaccharide by using copper nanoparticles and is loaded with bioactive substances by using gold nanoparticles.
In one embodiment, the bioactive agent is ovalbumin.
In one embodiment, the nano vaccine adjuvant enhances immune response by modulating MDA 5-IFN-alpha axis signaling pathway.
The invention also provides application of the nano vaccine adjuvant in preparation of a medicament for improving the release force of dendrobium polysaccharide.
Compared with the prior art, the invention has the beneficial effects that:
the nanometer vaccine adjuvant takes PBAE-G-B-PEG-SS-PEI polymer modified by nanometer calcium oxide as a core shell, the core embeds dendrobe polysaccharide macromolecules and OVA, the PBAE-G-B-PEG-SS-PEI polymer is modified by the nanometer calcium oxide to increase the hydrophilicity of the polymer, and the dendrobe polysaccharide can be quickly released from NP due to the conversion of the polymer from hydrophobicity to hydrophilicity.
The nano vaccine adjuvant P-CaO of the invention 2 The antigen specific IgG level in the immune serum of mice in the group of @ Au @ OVA @ Cu @ DHPs is obviously higher than that in the group of normal saline, which indicates that P-CaO 2 The use of @ Au @ OVA @ Cu @ DHPs as vaccine adjuvants can enhance immune response; in addition, P-CaO 2 The nanoparticle of @ Au @ OVA @ Cu @ DHPs has stable positive charge, can electrostatically adsorb mRNA with negative charge, and can be used with different dosage of P-CaO 2 The antigen specific antibody IgG is measured after the vaccine compound of @ Au @ OVA @ Cu @ DHPs/mRNA is immunized, and the 3 doses can induce the increase of SARS-Cov-2RBD IgG in serum, so that the P-CaO of the invention 2 The @ Au @ OVA @ Cu @ DHPs can become a high-efficiency vaccine carrier. In addition, the invention explores P-CaO 2 The immune enhancement mechanism of @ Au @ OVA @ Cu @ DHPs shows that P-CaO 2 @ Au @ OVA @ Cu @ DHPs enhance immune responses by modulating the MDA5-IFN- α axial signaling pathway.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of PBAE-G-B-PEG-SS-PEI of the present invention.
FIG. 2 shows P-CaO according to the present invention 2 The synthesis and characterization results of @ Au @ OVA @ Cu @ DHPs, wherein FIG. 2A is P-CaO 2 Schematic synthesis of @ Au @ OVA @ Cu @ DHPs; FIG. 2B and FIG. 2C are Cu @ DHPs, au @ OVA @ Cu @ DHPs and CaO, respectively 2 @ Au @ OVA @ Cu @ DHPs and P-CaO 2 SEM and EM images of nanoparticles of @ au @ ova @ cu @ dhps; FIG. 2D shows P-CaO 2 Nanoparticle element of @ Au @ OVA @ Cu @ DHPsAnalyzing a map; FIG. 2E particle size distribution; FIG. 2F is an analysis of aqueous solutions for P-CaO by DLS 2 PDI of @ Au @ OVA @ Cu @ DHPs nanoparticles; FIG. 2G shows P-CaO in different solvents 2 Zeta potential of the nanoparticle of @ Au @ OVA @ Cu @ DHPs; FIG. 2H and FIG. 2I are P-CaO, respectively 2 The DHPs-EE (%) and OVA-EE (%) of the @ Au @ OVA @ Cu @ DHPs nanoparticles have obvious difference (p) between different upper standard columns<0.05). Data represent mean ± SEM, n =3.
FIG. 3 shows P-CaO according to the present invention 2 Stability of @ Au @ OVA @ Cu @ DHPs in storage at 4 deg.C, where FIG. 3A is P-CaO 2 SEM images of @ Au @ OVA @ Cu @ DHPs at 0d, 7d, 14d, and 28 d; FIG. 3B is a graph of particle size, PDI, zeta potential, and zeta potential of nanoparticle suspensions measured over 28 days; FIG. 3C and FIG. 3D are graphs showing the measurement of P-CaO within 24 days, respectively 2 OVA release rate (%) and DHPs release rate (%) of the nanoparticle suspension of @ Au @ OVA @ Cu @ DHPs.
FIG. 4 shows P-CaO in accordance with the present invention 2 Cytotoxicity and macrophage uptake of antigen for @ Au @ OVA @ Cu @ DHPs, where FIG. 4A is macrophage with DHPs and P-CaO 2 Incubation activity of @ Au @ OVA @ Cu @ DHPs for 48 h; FIG. 4B shows OVA-FITC, PBAE-G-PEG-SS, and P-CaO 2 @ Au @ OVA @ Cu @ DHPs.
FIG. 5 shows P-CaO in accordance with the present invention 2 Activation of macrophage and P-CaO by @ Au @ OVA @ Cu @ DHPs 2 Effects of the transcriptome pathway activated by @ Au @ OVA @ Cu @ DHPs, wherein FIG. 5A and FIG. 5B are the expression of CD86+ and MHCII + on macrophages, respectively; FIGS. 5C and 5D are levels of cytokines TNF- α and IL-6 secreted by macrophages.
FIG. 6 shows P-CaO in accordance with the present invention 2 Examination of potential toxicity of @ Au @ OVA @ Cu @ DHPs in vivo, wherein FIG. 6A is H of mice inoculated with heart, lung, liver, spleen and kidney on day 35 after second immunization&E staining, scale bar: 40 μm; fig. 6B is serum biochemical (ALT, AST, ALP, LDH, BUN) levels of vaccinated mice on day 35 after the second immunization, with mean ± SEM showing no significant difference between n =3,n = groups.
FIG. 7 shows P-CaO according to the present invention 2 The CLSM profile and induced immune response profile of @ Au @ OVA @ Cu @ DHPs/mRNA vaccine complex, wherein FIG. 7A is the CLSM profile of the vaccine complex; FIG. 7B is an immune responseSchematic diagram of the experimental procedure; FIG. 7C shows that a-fbar significantly differs in histograms of different letters (P) for determination of RBD-specific IgG titer at given time points<0.05). Data are expressed as mean ± SEM, n =5.
FIG. 8 shows the levels of Th1 and Th2 cytokines in the serum of an immunized mouse detected by ELISA of the present invention, in which FIGS. 8A-8B show Th1 cytokines IFN-. Gamma.and TNF-. Alpha.respectively; figures 8C-8D are levels of the Th2 cytokines IL-4 and IL-6, respectively, with significant differences in histograms for the different letters (P < 0.05), (mean ± SEM, n = 4).
FIG. 9 shows P-CaO in accordance with the present invention 2 @ Au @ OVA @ Cu @ DHPs/mRNA stimulate cellular responses through MDA 5-IFN-. Alpha.Axis signaling pathway, where FIGS. 9A and 9B are P-CaO 2 Immunoblot analysis of MDA5-IFN- α following treatment of macrophages with @ Au @ OVA @ Cu @ DHPs/mRNA. The histograms of different letters differ significantly (P)<0.05). (mean ± SEM, n = 4).
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the 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 conventional methods unless otherwise specified.
Example one
1.1 materials
Dendrobe polysaccharides (DHPs, 95%, CY 200618) were obtained from maruzji, professor of agriculture university in south river. Anhydrous calcium chloride (CaCl) 2 ) Sodium hydroxide (NaOH) and hydrogen peroxide (H) 2 O 2 30%) from the Beijing chemical plant (Beijing, china). Copper chloride pentahydrate (CuSO) 4 ·5H 2 O), chloroauric acid was purchased from guangdong chemicals ltd, china. Succinylidinesuccinic acid-polyethylene glycol-COOH (SS-PEG-COOH, NOR-N-0027) was purchased from Ciceriz Biotech Ltd. 3- [4, 5-Dimethylthiazol-2-yl ]]2, 5-Diphenyltetrabromazole (MTT), RPMI 1640, fetal Bovine Serum (FBS), 0.25% pancreatin-EDTA, JC-1 probe, annexin V FITC/PI kit purchased from Saimer Feishel technology (Shanghai, china). Rhod-2 AM and MitoTracker Red were from Yeasen (Shanghai, china). 2',7' -dichlorofluorescein bisAcetate (DCFH-DA) was purchased from Bestbio (Shanghai, china). Alizarin red is produced by Yunaye (shanghai, china). The mouse TNF-alpha enzyme linked immunosorbent assay kit and the mouse IFN-gamma enzyme linked immunosorbent assay kit are purchased from Tianjin anaerobic biotechnology, inc. in China. Ovalbumin (OVA) was obtained from Sigma-Aldrich (MO, USA). All other reagents were of analytical grade.
1.2 FT-IR, NMR spectra and molecular morphology of DHPs
1D and 2D NMR spectra were acquired on 500MHz and 125MHz Bruk ARX500 spectrometers (Bruk, reinstein, germany), respectively. 2mg of dendrobii polysaccharide was completely dissolved in 0.5mL of D at 25 deg.C 2 And (4) in O. The spectra were observed by scanning 60000 times at 25 ℃. Data processing was performed using standard Bruker Topspin-NMR software.
DHPs samples were added to KBr and pressed into 1mm pellets for FT-IR measurements. The ASP spectra were recorded at 400-4000cm using an FT-IR spectrometer (Nicolet NEXUS 670) -1 In the frequency range of (c).
The molecular morphology of DHPs was observed using a FEI Quanta 250feg Scanning Electron Microscope (SEM). The samples were sputtered with a Crititon 108auto sputter coater with Pt and the images were observed at a voltage of 3.0kV for 800 and 1000 times high vacuum.
Synthesis of 1.3Au @ OVA @ Cu @ DHPs
5ml CuSO 4 ·5H 2 An aqueous O solution (10 mg/mL) and an aqueous DHPs solution (10 mL) were mixed and stirred at room temperature for 5min, and an aqueous NaOH solution (1M) was added to the above solution to adjust the pH to 10. After that, the solution was continuously stirred for 24 hours. The product (Cu @ DHPs) was collected by centrifugation and washed three times with deionized water. And finally collecting by freeze drying.
10mL of an aqueous solution of Cu @ DHPs (10 mg/mL) and 10mL of an aqueous solution of OVA were mixed and stirred at room temperature for 5 minutes, and an aqueous solution of sodium citrate was added to the above solution, and the mixture was stirred at 150 ℃ for 30 minutes. To the solution was added aqueous sodium chloride solution and stirring was continued for 15min. The product (Au @ OVA @ Cu @ DHPs) was collected by centrifugation and washed 3 times with deionized water. And finally collecting by freeze drying.
1.4CaO 2 Synthesis of @ Au @ OVA @ Cu @ DHPs
Taking 5ml of sodium acetateAqueous solution (5 mg/mL), 2.5mL CaCl 2 The aqueous solution (0.1M) and 2.5mL of an aqueous solution of Au @ OVA @ Cu @ DHPs were mixed and stirred at room temperature for 5min, and 5mL of an aqueous solution of NaOH (0.25M) was further added and stirred for 5min. Then 200. Mu.L of H 2 O 2 (30%) was added dropwise and stirred at room temperature for 30min. The product (CaO) was collected by centrifugation 2 @ Au @ OVA @ Cu @ DHPs) washed three times with deionized water. And finally collecting by freeze drying.
1.5 Synthesis of PBAE-G-B-PEG-SS-PEI
PBAE-G-B-PEG-SS-PEI conjugates were synthesized as shown in FIG. 1. The process can be divided into three steps: 1) Amino group of PBAE-G and DSPE-PEG 2000 Amidation reaction occurs between carboxyl groups of the-NHS to obtain PBAE-G-B; 2) Carrying out amidation reaction between amino of PBAE-G-B and carboxyl of SS-PEG-COOH to obtain PBAE-G-B-PEG-SS; 3) Amidation reaction is carried out between amino of PBAE-G-B-PEG-SS and carboxyl of polyetherimide (Cy5.5-PEI) with fluorescent dye (1.8 KD), and the polyetherimide is introduced to increase hydrophobicity of PBAE-G-B-PEG-SS, so as to obtain the PBAE-G-B-PEG-SS-PEI conjugate.
1.6P-CaO 2 Synthesis of @ Au @ OVA @ Cu @ DHPs
20mg of CaO 2 @ Au @ OVA @ Cu @ DHPs were added to a PBS (pH = 8.0) solution containing 200mg of PBAE-G-B-PEG-SS-PEI conjugate. The mixed solution was stirred continuously for 8h, dialyzed (cut-off molecular weight, MWCO 4500 Da) in PBS solution (pH = 7.4) and the organic solvent was removed. The product (P-CaO) was collected by centrifugation 2 @ Au @ OVA @ Cu @ DHPs) washed three times with deionized water. And finally collecting by freeze drying.
1.7 characterization of the nanoparticles
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 (Hydro 2000Mu, malvern Instruments, UK). The morphology of the nanoparticles was observed by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).
An improved microcolumn centrifugation method is adopted.
The calculation formula of the loading efficiency is as follows:
loading efficiency of DHPs = (1-Q) 1 /Q 2 )×100％，
Wherein: q 1 Is the number of idle dhps, Q 2 Is P-CaO 2 Total number of DHPs in the pause of @ Au @ OVA @ Cu @ DHPs.
OVA loading efficiency = (1-Q) 3 /Q 4 )×100％，
Wherein: q 3 Is the amount of free OVA, Q 4 Is P-CaO 2 Total amount of OVA in suspension of @ Au @ OVA @ Cu @ DHPs.
1.8 from P-CaO 2 Determination of ability to Release DHPs and OVA from @ Au @ OVA @ Cu @ DHPs
1mg of P-CaO 2 @ Au @ OVA @ Cu @ DHPs were dispersed in 2mL of water at different times and stirred for 12h. Centrifuging and collecting supernatant; the concentrations of DHPs and OVA in serum were determined by the phenol-sulfuric acid method.
1.9 stability of nanoparticles
The nanoparticle suspension was kept at 4 ℃ and the stability of the nanoparticles was examined. Particle size, PDI and Zeta potential were evaluated at 7d, 14d, 21d and 28d, respectively. The nanoparticles were measured for DHPs-EE (%) and OVA-EE (%) at the same time interval over 28 days. All experiments were measured in 3 replicates.
1.10 isolation of mouse peritoneal macrophages
ICR mice (6 weeks old) produced mouse peritoneal macrophages. Mice were injected intraperitoneally with 1mL 6% starch broth for 3 consecutive days. Macrophages were collected and their peritoneal cavity was lavaged twice with PBS. The separated cells were collected, centrifuged at 1500rpm for 8min, and suspended in complete medium at a cell density of 2X 105 cells/mL. Subsequently, the cell suspension was subjected to a humidified atmosphere (37 ℃,5% CO) 2 ) After culturing for 4h, the nonadherent cells are removed by washing with PBS, and fresh whole culture solution is added. The macrophages obtained were used for the next experiment.
1.11 macrophage viability Studies
The cell viability of peritoneal macrophages was assessed using the MTT method. The cells were seeded in 96-well plates at 100. Mu.L/well, and then 100. Mu.L of P-CaO was added 2 @ Au @ OVA @ Cu @ DHPs nanoparticles and free DHPs were diluted at different concentrations (0-500. Mu.g/mL). Cell groups and complete medium served as cell controls. In a humidified incubator (37 ℃, 5)％CO 2 ) After 48h of medium incubation, 30. Mu.L of MTT (5 mg/mL) was added, after 4h of incubation, the medium was removed and 100. Mu.L of DMSO was added to each well. The absorbance was measured at 570nm with a microplate reader (Thermo, USA). Cell viability was calculated as the ratio of absorbance of the experimental group to the control group. All experiments were repeated four times.
1.12 flow cytometry detection of MHCII and CD86
Expression of the surface markers MHCII and CD86 was detected by flow cytometry. Adding P-CaO 2 @ Au @ OVA @ Cu @ DHPs, free DHPs at a concentration of 30. Mu.g/mL, were co-cultured with macrophages for 48h, and then pancreatin-EDTA solution (containing phenol red) was added to the plate, centrifuged at 4000rpm for 5min, and labeled with anti-MHCII-FITC and anti-CD 86-PE in the dark for 30min. PBS was washed 3 times and flow cytometry detected the expression of MHCII and CD86 on macrophages.
1.13 secretion of cytokines by macrophages
Adding P-CaO 2 Culturing the @ Au @ OVA @ Cu @ DHPs and free DHPs with the concentration of 30 mu g/mL with macrophages for 48h, collecting cell culture supernatant after incubation, and detecting the levels of the cytokines TNF-alpha and IL-1 beta by adopting an ELISA kit, RT-PCR and Western Blot. Optical density (o.d.) was determined using a microplate reader at 450nm, and all experiments were measured in three replicates.
1.14 uptake Capacity of macrophages
With P-CaO 2 Mixing solution of @ Au @ OVA @ Cu @ DHPs with OVA-FITC (green), PBAG-G-PEG-SS (red) in a humidified incubator (37 deg.C, 5% 2 ) Incubate in 24-well plates for 12 hours. After incubation, cells were washed multiple times with PBS and stained with Hoechst 33342 for 10 minutes. The cells were washed several times with PBS to remove excess dye. Finally, the fluorescence image was recorded with an inverted microscope.
1.15 animals and Vaccination
1.15.1 preparation of SARS-CoV-2RBD mRNA vaccine
Reference for the preparation of SARS-CoV-2RBD mRNA (Ren et al, 2021). P-CaO 2 The @ Au @ OVA @ Cu @ DHPs/mRNA complex is prepared by mixing a certain amount of mRNA stock solution and different amounts of P-CaO in sterile distilled water 2 @ Au @ OVA @ Cu @ DHPs were freshly prepared. After gentle vortexing, incubate 30min at room temperatureAnd (4) forming the particles.
1.15.2 animal immunization
6-8 week-old female BALB/c mice were randomly divided into 6 groups (5 mice per group), and P-CaO was used for each group 2 @ Au @ OVA @ Cu @ DHPs/mRNA composite vaccine (5. Mu.g, 10. Mu.g, 30. Mu.g mRNA/mouse, N/P = 32) was injected intramuscularly 3 times, 1 time every 14 days. The negative control group was administered with an equal amount of physiological saline, 30. Mu.g of mRNA and PVES at the same time. Blood was collected from orbital veins 14 days and 28 days after the first immunization, respectively. The collected blood was centrifuged at 4000rpm to separate serum (30 minutes, 4 ℃). Serum was stored at-20 ℃ as subsequent SARS-CoV-2RBD specific IgG.
1.16 serum antibody evaluation
ELISA kit detects SARS-CoV-2RBD specific IgG antibody. ELISA kits were used to detect the titer of SARS-CoV-2 RBD-specific IgG according to the instructions. Briefly, 10-fold dilutions of serum were started from 1. After washing five times with the washing buffer, goat anti-mouse IgG labeled with horseradish peroxidase (HRP) was added to the petri dish and incubated at room temperature for 1 hour. The plates were then washed 5 times with wash buffer, developer solution was added and incubated for 20 minutes at room temperature. Absorbance at 450nm was read using a microplate reader. Endpoint titers were defined according to the manufacturer's instructions.
1.17 intracellular cytokine staining assay
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 isolated. Mouse splenocytes were placed in 12-well plates (1X 106 cells/well) and stimulated with peptide pools (2. Mu.g/mL single peptides) for 2h, golgiplug (BD Biosciences) was added at a final concentration of 1. Mu.L/mL, incubated for 4h, the cells collected and stained with anti-cd 4 and anti-cd 8. Alpha. Surface markers (Biolegend). Subsequently, the cells were fixed and permeabilized in permeation buffer (BD Biosciences) and stained with anti-ifn-gamma and anti-il-4 (Biolegend). Flow cytometry was performed using a BD FACSAria II flow cytometer and data was analyzed using FlowJo 10.0.
1.18 measurement of cytokines in serum
The concentrations of IFN-. Gamma.TNF-. Alpha.IL-4 and IL-6 cytokines in the serum were measured on days 21 and 28 after the initial inoculation using the Ready-to-use Sandwich ELISA kit according to the manufacturer's instructions. Optical Density (OD) was measured at 450nm and all experiments were repeated four times.
1.19 statistical analysis
All values are expressed as mean ± standard error of the mean (SEM). The multiple range test using Duncan and LSD is statistically significant for assessing differences. Probability values (p) less than 0.05 are considered statistically significant.
2 results and discussion
2.1P-CaO 2 Synthesis and characterization of @ Au @ OVA @ Cu @ DHPs.
As shown in fig. 2A, the Nanoparticles (NPs) of the present invention have a core-shell structure, and the core can encapsulate dendrobe polysaccharide macromolecules and OVA. The PBAE-G-B-PEG-SS-PEI polymer is modified by nano calcium oxide to increase the hydrophilicity, and the conversion of the polymer from hydrophobicity to hydrophilicity enables the dendrobium polysaccharide to be quickly released from NP.
It can be seen from FIGS. 2B and 2C that the P-CaO is well dispersed 2 @ Au @ OVA @ Cu @ DHPs exhibit a spherical morphology with a diameter of about 150 nm. Scanning Electron Microscope (SEM) results show that the DHPs and OVA coated by the core-shell bronze are spherical, the surface is slightly uneven, and the P-CaO 2 The @ Au @ OVA @ Cu @ DHPs nanoparticles are also approximately spherical and have slight unevenness, and the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM) clearly show P-CaO 2 The sheet-like morphology of @ Au @ OVA @ Cu @ DHPs. As can be seen from FIG. 2D, the individual P-CaO 2 The results of element mapping of @ Au @ OVA @ Cu @ DHPs revealed that dense C (red) and O (lake blue) were distributed around the Au (violet) core, confirming P-CaO 2 @ Au @ OVA @ Cu @ DHPs are uniformly distributed.
P-CaO as shown in FIG. 2E 2 Average particle size of @ Au @ OVA @ Cu @ DHPs is slightly larger than CaO 2 @Au@OVA@Cu@DHPs，P-CaO 2 The average particle size of @ Au @ OVA @ Cu @ DHPs was 157.2. + -. 2.21nm. PDI were all below 0.3, indicating a narrow size distribution (fig. 2F).
P-CaO was investigated by measuring zeta potential (dissolution rate difference) 2 @ Au @ OVA @ Cu @ DHPs sodiumStability of rice grains, as shown in fig. 2G. P-CaO 2 Zeta-potentials of @ Au @ OVA @ Cu @ DHPs nanoparticles in water (11.90. + -. 0.71 mV), 10% FBS (12.30. + -. 1.45 mV) and PBS (11.73. + -. 1.11 mV) were all positively charged. Furthermore, there was no difference in zeta potential between day 1 and day 35 (p)>0.05)。
As shown in FIG. 2H, cu @ DHPs, au @ OVA @ Cu @ DHPs, caO 2 @ Au @ OVA @ Cu @ DHPs and P-CaO 2 The DHPs entrapment rate of the @ Au @ OVA @ Cu @ DHPs nanoparticles is 55.7 +/-1.31%, 54.17 +/-2.60%, 50.53 +/-1.59% and 47.8 +/-1.11%. As shown in FIG. 2I, au @ OVA @ Cu @ DHPs, caO 2 @ Au @ OVA @ Cu @ DHPs and P-CaO 2 The OVA entrapment rate of the @ Au @ OVA @ Cu @ DHPs nanoparticles is 62.83 +/-0.81%, 56.03 +/-2.09% and 53.73 +/-0.52%. These results represent P-CaO 2 @ Au @ OVA @ Cu @ DHPs were successfully prepared.
2.2 stability of the nanoparticles
To evaluate P-CaO 2 Stability of nanoparticle suspension of @ Au @ OVA @ Cu @ DHPs, morphology of the nanoparticles was measured at 4 ℃ with Transmission Electron Microscopy (TEM) for 4 weeks. P-CaO, as shown in FIG. 3A 2 The nano-particles of @ Au @ OVA @ Cu @ DHPs have little change in morphology. As shown in FIG. 3B, P-CaO was present over a 28 day period 2 The average size of the nanoparticles of @ Au @ OVA @ Cu @ DHPs varies from 172.5nm to 186.2 nm. The PDI of the nanoparticles has no obvious change or deviation within 28 days and is lower than 0.3, which indicates that the particle size distribution of the nanoparticles is uniform within 28 days. The zeta potential of the nanoparticles was also unchanged within 28 days.
2.3 from P-CaO 2 Ability of nanoparticles of @ Au @ OVA @ Cu @ DHPs to release DHPs and OVA
As shown in FIG. 3C, P-CaO in the cell-simulated environmental fluid 2 The OVA coated by the @ Au @ OVA @ Cu @ DHPs nano particles is slowly released on the 1 st day, and the accumulated release amount is about 5.73 +/-0.06%. However, in a cell-simulated environment, from 1 to 16 days, P-CaO 2 The nano-particles of @ Au @ OVA @ Cu @ DHPs release OVA rapidly, and the cumulative release amount is about 42.97 +/-0.20%. From 16 to 36d, the OVA release rate was increased again, and the cumulative release amount was about 47.15. + -. 0.56%. This is probably due to the degradation of PBAE-G-B-PEG-SS-PEI conjugates and CaO 2 The damage of emulsion structure causes OVA to be released in 16 th to 36 th daysDue to the acceleration. The cumulative release result shows that P-CaO 2 The nano-particles of @ Au @ OVA @ Cu @ DHPs have good controlled release effect. At the same time, P-CaO 2 In vitro release of DHPs from the nanoparticle of @ Au @ OVA @ Cu @ DHPs As shown in FIG. 3D, DHPs were released slowly on the first day and then rapidly. The cumulative amount of DHPs released was about 57.25. + -. 0.12% at 36 days.
2.4P-CaO 2 Cytotoxicity of nanoparticles of @ Au @ OVA @ Cu @ DHPs and uptake of antigen by macrophages
Research on DHPs and P-CaO by MTT method 2 Cytotoxicity of particles of @ Au @ OVA @ Cu @ DHPs. Macrophages were exposed to different concentrations of GO and GO-LNT for 48 hours. DHPs and P-CaO 2 @ Au @ OVA @ Cu @ DHPs were non-toxic to macrophages at 30. Mu.g/mL (FIG. 4A). Therefore, a concentration of 30. Mu.g/mL was chosen for subsequent experiments.
The ability of macrophages to efficiently take up antigen is a prerequisite for antigen presentation and for the stimulation of immune responses. Macrophages and neutrophils play important roles in both innate and adaptive immunity, constituting the first line of defense against pathogen invasion. To study the uptake of antigen by macrophages, OVA-FITC, PBAE-G-PEG-SS, and P-CaO were observed using a confocal laser microscope 2 Ingestion of @ Au @ OVA @ Cu @ DHPs. FIG. 4B shows higher internalization of OVA-FITC compared to OVA-FITC alone. The results show P-CaO 2 @ Au @ OVA @ Cu @ DHPs induce macrophages to efficiently take up antigen.
2.5 macrophage surface molecule expression level and cytokine secretion
Macrophages are activated after phagocytosis and provide antigens to T lymphocytes, thereby activating an adaptive immune response. In addition, macrophages also play a key role in humoral and cellular immunity. Following antigen uptake by cells, macrophages highly express costimulatory molecules. CD86 + And MHCII + Is associated with presentation of exogenous antigens to T cells and promotion of T cell activation. For detecting P-CaO 2 The effect of @ Au @ OVA @ Cu @ DHPs on macrophage activation, and flow cytometry is adopted to detect co-stimulatory molecule MHCII + And CD86 + Expression of (2). As shown in FIGS. 5A and 5B, compared with the control groupSpecific, P-CaO 2 MHCII of @ Au @ OVA @ Cu @ DHPs group + And CD86 + Expression is significantly upregulated (p)<0.05 No difference from LPS group (p)>0.05)。P-CaO 2 Group CD86 of @ Au @ OVA @ Cu @ DHPs + The expression is obviously higher than that of other groups (p)<0.05)。
Cytokines secreted by immune cells, particularly macrophages, play a key role in the initiation and regulation of immune responses, which are essential for the clearance of infected cells and pathogens. With P-CaO 2 In vitro macrophage stimulation with @ Au @ OVA @ Cu @ DHPs, ELISA was performed to determine the levels of IL-6 and TNF- α in the supernatants. P-CaO as shown in FIGS. 5C and 5D 2 The IL-1 beta and TNF-alpha secretion induced by @ Au @ OVA @ Cu @ DHPs is higher than that induced by other groups (P)<0.05 and P<0.01)。
2.6P-CaO 2 In vivo potential toxicity of @ Au @ OVA @ Cu @ DHPs
To investigate P-CaO 2 The toxicity of GO-LNT/OVA on main organs is evaluated if @ Au @ OVA @ Cu @ DHPs are safe as vaccine adjuvants. Taking the tissue of heart, lung, liver, spleen and kidney, H&And E, staining detection. Compared to the PBS group, the tissue structure was normal in the experimental group, with no significant inflammation and injury (fig. 6B). In order to further explore the potential toxicity of GO-LNT/OVA in vivo, the present invention performed biochemical serum assays in mice. Liver function index (ALT, AST, ALP), myocardial function index (LDH), and renal function index (BUN) were measured. ALT, AST and ALP levels in serum are important indexes for detecting liver function. Significant elevation of AST, ALT, ALP levels is indicative of liver damage. As the blood urea nitrogen excretion capacity decreases, the kidneys are damaged, resulting in increased levels of BUN in the kidneys. Lactate dehydrogenase is a glycolytic enzyme, an increase in activity of which is commonly observed in diseases affecting the heart muscle or liver. P-CaO 2 The ALT, AST, ALP, LDH and BUN groups were not significantly changed and remained within the normal range (FIG. 6A). These results show that: P-CaO 2 @ Au @ OVA @ Cu @ DHPs are safe as vaccine adjuvants.
2.7P-CaO 2 Immune response induced by vaccine complex of @ Au @ OVA @ Cu @ DHPs
P-CaO was observed by CLSM as shown in FIG. 7A 2 Vaccine complex of @ Au @ OVA @ Cu @ DHPs. SARS-CoV-2RBD mRNA in vaccine compositionThe product showed green fluorescence. In addition, P-CaO was observed in the vaccine complex 2 Red fluorescence of @ Au @ OVA @ Cu @ DHPs.
To further verify P-CaO 2 Use of @ Au @ OVA @ Cu @ DHPs as mRNA vaccine adjuvant and for inducing immune response in vivo with different doses of P-CaO 2 5 groups of mice were immunized with the @ Au @ OVA @ Cu @ DHPs/mRNA vaccine complex (5. Mu.g, 10. Mu.g and 30. Mu.g/mouse) (n = 5). Mice were immunized 0 during day time, boosted at day 14, sacrificed at days 21, 28, 35, and 42, and serum samples were collected for antibody and cytokine analysis (fig. 7B). Spleens were harvested on day 28 for histopathological analysis. Mice injected with PBS were used as control groups. The titer of RBD-specific antibodies was measured by ELISA to assess humoral immune responses (fig. 7C). The results show that three doses of P-CaO 2 The @ Au @ OVA @ Cu @ DHPs/mRNA complex produced significant antibody titers 21 days after the first immunization. After boosting, antibody levels rapidly increased. The titer of the mice immunized with the high dose of the RBD-specific antibody is obviously higher than that of the mice immunized with the low dose. Mean endpoint titers rose to after the third immunization in the 30. Mu.g group>10 5 Compared with the 10 mu g group and the 5 mu g group, the increase is 1.5 times and 5.2 times respectively. Therefore, we chose a dose of 30 μ g to immunize mice in the next study. The levels of antibody isotypes IgG1, igG2a and IgG2b were detected as shown in FIG. 7C. The results agree well with the assumptions.
2.8 cytokine levels in serum
The cytokine concentration in serum was measured by ELISA at 21, 28 and 35 days after the first inoculation. P-CaO compared to the other groups 2 Significant increase in Th1 type cytokine (IFN-gamma and TNF-alpha) levels induced by @ Au @ OVA @ Cu @ DHPs/mRNA (P<0.05 (fig. 8A and 8B). TNF- α plays a key role in cellular immune processes and can promote Th1 responses. IL-4 and IL-6 secretion amount P-CaO 2 The change trend of @ Au @ OVA @ Cu @ DHPs/mRNA is similar to IFN-gamma and TNF-alpha and is obviously higher than that of other groups (P)<0.05 (FIG. 8C, FIG. 8D). These results indicate P-CaO 2 The secretion of Th1 and Th2 cytokines was induced by @ Au @ OVA @ Cu @ DHPs/mRNA, consistent with the above-described antibody response results.
2.9P-CaO 2 Passage of @ Au @ OVA @ Cu @ DHPs/mRNAMDA 5-IFN-alpha axis signaling pathway stimulates cellular responses
The SARS-CoV-2 spike protein binds to and stimulates TLR2 and TLR4 signaling. In addition, the immune response to seasonal influenza and other non-adjuvanted vaccines is controlled by the microbiota through the TLR5 pathway. The BNT162b2 mRNA vaccine co-developed by Peverine and BioNTech, USA, stimulates CD8+ T cell response through MDA 5-IFN-alpha axis.
The invention passes through P-CaO 2 Stimulation of CD8 by @ Au @ OVA @ Cu @ DHPs/mRNA + T cell detection and MDA 5-IFN-alpha axis signal pathway related gene, and P-CaO 2 The action and mechanism of @ Au @ OVA @ Cu @ DHPs/mRNA. P-CaO as shown in FIGS. 9A and 9B 2 The protein expression levels of MDA5, cGAS, STING and IFN-gamma of LPS group and Control group can be obviously improved by @ Au @ OVA @ Cu @ DHPs/mRNA. In addition, P-CaO 2 @ Au @ OVA @ Cu has a structure similar to P-CaO 2 With the similar action of @ Au @ OVA @ Cu @ DHPs, mRNA can increase the protein expression levels of MDA5, cGAS, STING and IFN-gamma, which is similar to that of LPS group and Control group. The above results show P-CaO 2 @ Au @ OVA @ Cu @ DHPs/mRNA stimulate cellular responses through the MDA 5-IFN-. Alpha.axis signaling pathway.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.
Claims (6)
1. The nanometer vaccine adjuvant is characterized in that the nanometer vaccine adjuvant is a core-shell structure which is formed by taking a cationic polymer modified by nanometer calcium oxide as a core shell and embedding dendrobium polysaccharide and bioactive substances in the core.
2. The nano-vaccine adjuvant of claim 1, wherein the cationic polymer is a PBAE-G-B-PEG-SS-PEI polymer.
3. The nano vaccine adjuvant according to claim 1, wherein the core of the nano vaccine adjuvant is loaded with dendrobium polysaccharide by using copper nanoparticles and is loaded with bioactive substances by using gold nanoparticles.
4. The nano vaccine adjuvant according to claim 1 or 3, characterized in that the bioactive substance is ovalbumin.
5. The nano vaccine adjuvant according to any one of claims 1 to 4, wherein the nano vaccine adjuvant enhances immune response by regulating MDA 5-IFN-alpha axis signal pathway.
6. The use of the nano vaccine adjuvant according to any one of claims 1 to 5 in the preparation of a medicament for improving the release force of dendrobium polysaccharides.
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