A vaccine adjuvant and preparation thereof

Abstract

The application belongs to the field of marine biotechnology, and particularly relates to natural polysaccharide-quaternized chitosan nanoparticles with immune stimulation and preparation and application thereof. The nanoparticles formed by natural polysaccharide with negative charge and quaternized chitosan with positive charge are obtained by polyelectrolyte complexation, the particle size is 316.4 nm, and the potential is 31.1 mV; the quaternized chitosan with positive charge and the natural polysaccharide with negative charge are mixed in a mass ratio of 6.25-10:1-4. After immunization of mice, the secretion amount of immunoglobulin IgG and immune factors in the mice is determined, which shows that the nanoparticles have good immune activity, thereby playing an effect in vaccine adjuvant. The application provides a method and guidance for the research on natural polysaccharide and chitosan derivatives as immune adjuvants in recent years.

Description

Technical Field

[0001] This invention belongs to the field of marine biotechnology, specifically relating to a vaccine adjuvant and its preparation method. Background Technology

[0002] The most basic function of adjuvants is to protect antigens and provide a certain degree of sustained release. Existing vaccine adjuvants are mostly aluminum salt adjuvants and white oil adjuvants, which have problems such as being difficult to degrade after injection and easily causing inflammation. Therefore, there is a need to develop a novel adjuvant that can exert its adjuvant effect while being degraded in vivo without causing harm to the body.

[0003] Natural polysaccharides are widely available, highly biocompatible, and exhibit excellent activity. They are also safe, non-toxic, and possess good biocompatibility. Nanoparticle research has been a hot topic in recent years, and using nanoparticles as drug carriers is an important research direction for many drugs in medicine. However, polysaccharides mostly exist in chain form, making them easily degraded after entering the body. Overly stable nanoparticles are not conducive to antigen release. Therefore, preparing polysaccharides into nanoparticles can slow down the degradation rate of polysaccharides and simultaneously achieve a sustained-release effect on antigens. Thus, the development of vaccine adjuvants based on natural polysaccharides has certain advantages. Summary of the Invention

[0004] In view of the above problems, the present invention provides a vaccine adjuvant and a method for preparing the same.

[0005] To achieve the above objectives, the technical solution adopted in this experiment is as follows:

[0006] A vaccine adjuvant, comprising nanoparticles formed by combining natural polysaccharide-quaternized chitosan with a polyelectrolyte, wherein the nanoparticles have a particle size of 316.4 nm and a potential of 31.1 mV; wherein the positively charged and negatively charged derivatives are mixed in a mass ratio of 6.25-10:1-4.

[0007] The natural polysaccharide is heparin; the quaternized chitosan is glycidyltrimethylammonium chloride chitosan with a molecular weight of 200 kDa.

[0008] An application of natural polysaccharide-quaternized chitosan nanoparticles obtained by a preparation method, wherein the natural polysaccharide-quaternized chitosan nanoparticles are used in the preparation of vaccine adjuvants with immunostimulatory effects.

[0009] An immunostimulant that encapsulates an antigen, wherein the vaccine adjuvant is the natural polysaccharide-quaternized chitosan nanoparticles as described in claim 1, wherein the polysaccharide and the antigen are mixed in a mass ratio of 0.5-2:1.

[0010] After mixing positively charged quaternized chitosan with the antigen, negatively charged natural polysaccharides are added, and an immunostimulant encapsulating the antigen is prepared through electrostatic adsorption.

[0011] The positively charged quaternized chitosan and antigen were magnetically stirred at 500-700 rpm for 5-10 minutes at room temperature. Then, the negatively charged polysaccharide was added and the stirring was continued for 20-40 minutes. Nanoparticles encapsulating the antigen were prepared by electrostatic adsorption. After filtration, the stimulant was obtained and stored at 4°C.

[0012] Furthermore, using the chitosan derivative nanoparticles, the results of mouse immunization experiments showed that the nanoparticles could effectively enhance the immunostimulatory activity of antigens and better promote the secretion of immunoglobulin IgG and IL-17 immune factors in mice. This indicates that the nanoparticles have the potential to serve as an immunostimulatory vaccine adjuvant and can exert a good immunoadjuvant effect.

[0013] Advantages of this invention:

[0014] 1. The vaccine adjuvant of the present invention uses negatively charged natural polysaccharides and positively charged chitosan derivatives to prepare nanoparticles without introducing cross-linking agents, thereby eliminating the toxic side effects of cross-linking agents and ensuring its safety in vivo.

[0015] 2. Using the immune adjuvant of this invention to load antigens for the determination of mouse immunization results, it was shown that the immune adjuvant of this invention can effectively load antigens and enhance the immunostimulatory effect of antigens, promoting the secretion of immunoglobulin IgG and IL-17 immune factors in mice. It has good application value in the preparation of highly efficient immune adjuvants without side effects. Attached Figure Description

[0016] Figure 1 The infrared spectrum is shown for the chitosan quaternary ammonium salt derivative obtained in Example 1 of this invention.

[0017] Figure 2 The following are characterization diagrams of the potential (A) and particle size (B) of the chitosan derivative nanoparticles obtained in Example 2 of the present invention; (A) is the potential diagram, and (B) is the particle size diagram.

[0018] Figure 3 This is a scanning electron microscope image of the negatively charged natural polysaccharide-chitosan derivative nanoparticles obtained in Example 2 of the present invention.

[0019] Figure 4 Figure 3 shows the mouse immunoglobulin IgG and immune factor IL-17 secreted after immunization obtained in Example 3 of the present invention; (A) is IgG1, (B) is IgG2, and (C) is IL-17. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings, and the scope of protection of the present invention is not limited to the following embodiments.

[0021] This invention uses negatively charged natural polysaccharides and positively charged quaternized polysaccharides as raw materials to prepare an immune adjuvant and determine its effect on the immune level of mice, ultimately obtaining negatively charged natural polysaccharide-quaternized chitosan nanoparticles with vaccine adjuvant function.

[0022] The specific method is as follows: Antigen-loaded nanoparticles are prepared by combining negatively charged natural polysaccharides and positively charged quaternized chitosan with a molecular weight of 200 kDa using a polyelectrolyte composite method. Nanoparticles with uniform particle size and stable potential are screened using a nanoparticle size analyzer. Animal experiments are then conducted to determine the immunogenicity of the nanoparticles after injection into mice, identifying immunogenic nanoparticles. The nanoparticles prepared under the above raw materials and conditions have no toxic side effects and can exert good dual effects of immunogenicity and as a carrier. This provides a certain method and guidance for the research on natural polysaccharides as vaccine adjuvants in recent years.

[0023] Example 1: Preparation of Quaternized Chitosan

[0024] 5g of chitosan with a molecular weight of approximately 200kDa and 10g of 2,3-epoxypropyltrimethylammonium chloride were taken. 70mL of distilled water was added, and the mixture was stirred in an 80℃ water bath at 200 rpm for 24 hours. The resulting reaction solution was dialyzed against distilled water in a dialysis bag for 72 hours, and then freeze-dried at -80℃ to obtain quaternized chitosan samples. The obtained chitosan quaternary ammonium salt was characterized by infrared spectroscopy, and the results are shown below. Figure 1 .Depend on Figure 1 As can be seen, the infrared spectrum at 1478 cm⁻¹ -1 The appearance of a new absorption peak indicates that the quaternary ammonium salt chitosan was successfully synthesized. Furthermore, the molecular weight of the synthesized quaternary ammonium salt chitosan is approximately 200 kDa.

[0025] Example 2: Preparation of negatively charged natural polysaccharide-quaternized chitosan nanoparticles

[0026] Reagents: Using double-distilled water as solvent, prepare the following solutions sequentially under aseptic conditions: 1.0 mg / mL chitosan quaternary ammonium salt solution a, 1.5 mg / mL negatively charged natural polysaccharide solution b, and 2.0 mg / mL standard antigen OVA solution m.

[0027] Take 5 mL of solution a and place it in a 25 mL beaker. Place the beaker in a magnetic stirrer at 300 rpm. At the start of stirring, add 2 mL of antigen solution m dropwise. After stirring for 10 min, add solution b dropwise. Continue stirring at the same rate for 30 min. Filter to obtain a nanoparticle solution, store at 4 °C, and obtain antigen-loaded nanoparticles (see [link to product]). Figure 2 and 3The physicochemical properties of the nanoparticles are shown in Table 1. The particle size of the nanoparticles was determined to be 316.4 nm and the potential was 31.1 mV.

[0028] The results of potential and particle size show that, in order to ensure the stability of nanoparticles and to better carry and release them in cells and subsequent animals, nanoparticles with a particle size of 316.4 nm, a potential of 31.1 mV, and a dispersion of 0.171 were obtained.

[0029] Table 1. Physicochemical properties of negatively charged natural polysaccharide-quaternized chitosan nanoparticles:

[0030] sample Dispersion PDI Potential (mV) Particle size (nm) Heparin-quaternized chitosan 0.171 31.1 316.4

[0031] Example 3: Effect of negatively charged natural polysaccharide-quaternized chitosan nanoparticles on immune levels. The nanoparticles prepared above, heparin-quaternized chitosan (HS-HACC-OVA), PBS (Control), negative control, and OVA (positive control) were used for detection.

[0032] 1) Effects of negatively charged natural polysaccharide-quaternized chitosan nanoparticles on the level of secreted immunoglobulin IgG in mice.

[0033] Immunoglobulin IgG plays a crucial role in the immune response by activating complement and neutralizing various toxins. It is also one of the most important antibody components in the body, with IgG1 and IgG2 being the most significant. The levels of these two antibodies largely indicate the levels of humoral and cellular immunity.

[0034] The Solarbio ELISA kit was used to determine the levels of IgG1 and IgG2 in the serum of mice after immunization. The measurement procedure was performed according to the kit instructions, and the specific method is as follows:

[0035] IgG1 content determination:

[0036] 1. 20 minutes before the experiment, take out the reagent kit and allow it to return to room temperature. Before adding the standards / samples, wash the plate twice and shake it dry.

[0037] 2. Add 100 μL of standard and test sample to each well, and add 50 μL of enzyme-labeled antibody working solution to each well. After sealing the plate, incubate overnight at room temperature (25±2℃) for 15 h with shaking at 400 rpm.

[0038] 3. After tapping and washing the plate 4 times, add 100μL of the chromogenic substrate to the reaction wells, seal the plate, and develop the color at room temperature (25±2℃) in the dark for 5-20 minutes.

[0039] 4. Add 50 μL of stop solution and immediately measure the OD value at 450 nm wavelength using a microplate reader (within 5 minutes).

[0040] IgG2 content determination:

[0041] 1. 30 minutes before the experiment, take out the reagent kit and allow it to return to room temperature. Before adding the standards / samples, wash the plate 3 times and shake it dry.

[0042] 2. Add 100 μL of standard and test sample to the reaction well, seal the plate and incubate at room temperature (25±2℃) with shaking for 120 min.

[0043] 3. After tapping and washing the plate 4 times, add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, and incubate at room temperature (25±2℃) with shaking for 60 min.

[0044] 4. After tapping and washing the plate 4 times, add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate and incubate at room temperature (25±2℃) with shaking for 30 min.

[0045] 5. After tapping and washing the plate 5 times, add 100 μL of the chromogenic substrate to the reaction wells, seal the plate, and incubate at room temperature (25±2℃) in the dark for 5-20 min.

[0046] 6. Add 50 μL of stop solution and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 minutes).

[0047] Test results IgG1 ( Figure 4 A) and IgG2 ( Figure 4 B) As shown in the bar chart, compared with the use of the antigen alone and the blank control group, the nanoparticles can promote higher levels of secretion of immunoglobulins IgG1 and IgG2 in mice.

[0048] 2) Effects of negatively charged natural polysaccharide-quaternized chitosan nanoparticles on the secretion of the mouse immune factor IL-17.

[0049] IL-17 is produced by CD4 + T cell secretion of IL-17 promotes the aggregation of inflammatory chemokines, acute-phase proteins, and neutrophils, thus playing a role in the body's immunity and autoimmune diseases. IL-17 activates the NOTCH signaling pathway in cells, participating in cell proliferation and regulating the expression of inflammatory cytokines. The IL-17 level in the serum of immunized mice was measured using an ELISA method, following the kit instructions.

[0050] 1. 30 minutes before the experiment, take out the reagent kit and allow it to return to room temperature. Before adding the standards / samples, wash the plate 3 times and shake it dry.

[0051] 2. Add 100 μL of standard and test sample to the reaction well, seal the plate and incubate at room temperature (25±2℃) with shaking for 120 min.

[0052] 3. After tapping and washing the plate 4 times, add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, and incubate at room temperature (25±2℃) with shaking for 60 min.

[0053] 4. After tapping and washing the plate 4 times, add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate and incubate at room temperature (25±2℃) with shaking for 30 min.

[0054] 5. After tapping and washing the plate 5 times, add 100 μL of the chromogenic substrate to the reaction wells, seal the plate, and incubate at room temperature (25±2℃) in the dark for 5-20 min.

[0055] 6. Add 50 μL of stop solution and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 minutes).

[0056] The measurement results are as follows Figure 4 As shown in the bar chart in section C, compared with the use of antigen alone and the blank control group, the nanoparticles can promote higher levels of secretion of the immune factor IL-17 in mice.

[0057] In summary, natural heparin can be used as a negatively charged polysaccharide and a positively charged substance to prepare nanoparticles. The prepared nanoparticles have a diameter of 316.4 nm and a potential of 31.1 mV, and are relatively stable. After animal immunization, compared with the use of antigen OVA alone and the control group, it can better promote the secretion of immunoglobulin IgG and immune factor IL-17 in mice. This indicates its potential as a vaccine adjuvant and provides a reference for addressing the problems of poor in vivo degradation and inflammation caused by current vaccine adjuvants.

Claims

1. A vaccine adjuvant, characterized in that: The vaccine adjuvant is a nanoparticle formed by a composite of a negatively charged natural polysaccharide and a positively charged quaternized chitosan with a polyelectrolyte. The nanoparticle has a particle size of 316.4 nm and a potential of 31.1 mV. The positively charged quaternized chitosan and the negatively charged natural polysaccharide are mixed in a mass ratio of 6.25-10:1-4. The negatively charged natural polysaccharide is heparin; the positively charged quaternized chitosan is glycidyltrimethylammonium chloride chitosan with a molecular weight of 200 kDa.

2. The application of the vaccine adjuvant according to claim 1, characterized in that: Application of negatively charged natural polysaccharides-positively charged quaternized chitosan nanoparticles in the preparation of vaccine adjuvants with dual functions of immunostimulation and carrier.

3. The application of the vaccine adjuvant according to claim 2, characterized in that: The vaccine adjuvant and antigen are mixed at a mass ratio of 0.5-2:

1.

4. The application of the vaccine adjuvant according to claim 3, characterized in that: After mixing positively charged quaternized chitosan with the antigen, negatively charged natural polysaccharides are added, and the antigen-loaded vaccine is prepared through electrostatic adsorption.

5. The application of the vaccine adjuvant according to claim 4, characterized in that: The positively charged quaternized chitosan and antigen were magnetically stirred at 500-700 rpm for 5-10 minutes at room temperature, and then the negatively charged natural polysaccharide was added and the stirring was continued for 20-40 minutes. The antigen-loaded vaccine was prepared by electrostatic adsorption, and the filtered solution was stored at 4°C.

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