阅读说明：本技术 一种壳聚糖纳米硒颗粒及其制备方法和在疫苗中的应用 (Chitosan nano-selenium particle, preparation method thereof and application thereof in vaccine ) 是由 周志昉 俞杭艳 汪楚卉 解云天 林汉 于 2021-01-19 设计创作，主要内容包括：本发明公开了一种壳聚糖纳米硒颗粒及其制备方法和在疫苗中的应用,其中所述壳聚糖纳米硒通过壳聚糖和亚硒酸钠等原料制备,其可实现对抗原和疫苗佐剂的高效包裹。该壳聚糖纳米硒颗粒具有极好的生物安全性。该壳聚糖纳米硒颗粒具有良好的物理稳定性、热稳定性,适用于疫苗工业化制备。该壳聚糖纳米硒颗粒作为免疫载体或佐剂,能够促进抗原呈递细胞对抗原的吞噬、处理和呈递,诱导产生更高水平的抗原特异性IgG抗体效价,在疫苗开发领域具有重要的应用价值。(The invention discloses chitosan nano selenium particles, a preparation method thereof and application thereof in vaccines, wherein the chitosan nano selenium is prepared from raw materials such as chitosan, sodium selenite and the like, and can realize efficient coating of antigens and vaccine adjuvants. The chitosan nano selenium particle has excellent biological safety. The chitosan nano selenium particle has good physical stability and thermal stability, and is suitable for the industrial preparation of vaccines. The chitosan nano selenium particle is used as an immune carrier or adjuvant, can promote phagocytosis, treatment and presentation of antigen by antigen presenting cells, induces and generates higher-level antigen-specific IgG antibody titer, and has important application value in the field of vaccine development.)

1. The chitosan nano selenium particle loaded with the antigen and the vaccine adjuvant is characterized in that chitosan with positive charges and sodium selenite are subjected to reduction reaction to form the chitosan nano selenium particle, the chitosan nano selenium particle is spherical, has a solid spherical core and has a particle size of 20-200 nm.

2. The chitosan nano-selenium particle according to claim 1, wherein the chitosan nano-selenium particle has a PDI of 0.1-0.4 and a Zeta potential of 15-40 mV.

3. The method for preparing chitosan nano-selenium particle according to claim 1 or 2, characterized by comprising the steps of: adding a sodium selenite solution into an acetic acid solution of chitosan, magnetically stirring at room temperature, adding an ascorbic acid solution for reaction, standing for aging, and dialyzing and purifying through a dialysis bag to obtain the chitosan nano selenium particles.

4. The method for preparing chitosan nano selenium particle according to claim 3, wherein the concentration ratio of sodium selenite to ascorbic acid is 2: 1-1: 20; the content of the ascorbic acid solution in the reaction solution is 0-0.50 mol/L; the concentration of the acetic acid solution of the chitosan is 0.1-5 mg/mL.

5. The method for preparing chitosan nano-selenium particle according to claim 3, wherein the reaction condition of the standing is: and keeping away from light, wherein the reaction temperature is 0-100 ℃.

6. The use of the chitosan nano selenium particle according to claim 1 or 2 or the chitosan nano selenium particle prepared by the preparation method of claim 3 or 4 as an antigen carrier or a vaccine adjuvant, characterized in that the chitosan nano selenium particle and a negatively charged antigen form a vaccine particle by means of electrostatic adsorption or chemical covalent coupling.

7. The use of chitosan nano selenium particle as antigen carrier or vaccine adjuvant according to claim 6, wherein the vaccine adjuvant is selected from one of ligand CpG-ODN of Toll-like receptor 9, ligand MPLA of Toll-like receptor 4, aluminum adjuvant.

8. The application of the chitosan nano selenium particle as an antigen carrier or a vaccine adjuvant according to claim 6, wherein the particle size of the vaccine particle is 1-1000 nm, preferably 20-300 nm.

9. The use of chitosan nano-selenium particles in the preparation of anti-tumor drugs according to claim 1 or 2, wherein the chitosan nano-selenium particles and chicken ovalbumin OVA form nanoparticles through electrostatic adsorption.

10. The application of the chitosan nano selenium particles in the preparation of antitumor drugs according to claim 9, wherein the particle size of the nanoparticles is 50-300 nm; PdI is 0.1-0.2; the Zeta potential is 15-35 mV.

Technical Field

The invention relates to the technical field of vaccine development, in particular to chitosan nano selenium particles, a preparation method thereof and application thereof in vaccines.

Background

Vaccine development has gone through several generations of technological updates. Live and inactivated attenuated vaccines are widely used to prevent severe viral diseases including yellow fever, measles and polio, but have the disadvantages of difficult culture, susceptibility to infection, and susceptibility to provoking an exaggerated immune response. Subunit vaccines, including proteins, polysaccharides or other pathogen components, such as influenza b, diphtheria, tetanus, acellular perforation, meningococci and pneumococci, also require adjuvants to enhance their immunogenicity and often undergo early degradation after exposure to hostile in vivo environments. Ribonucleic acid or deoxyribonucleic acid vaccines have a minimal risk of infection, are capable of eliciting an immune response against a particular pathogen, and are inexpensive; but face the problem of premature degradation of the molecule and the inability to convert into a functional immunogen.

To overcome these obstacles, an effective vaccine delivery system is needed. The development of a new generation of composite vaccine by adopting the nanotechnology is a good choice. The nanocarrier-based delivery system can protect vaccines from premature degradation and has good adjuvant properties that facilitate targeted delivery of immunogens to Antigen Presenting Cells (APCs). Vaccine antigens may be encapsulated in nanocarriers or modified on their surface. Encapsulation within the nanoparticle can protect antigens from premature protease degradation and allow for sustained release, while surface-adsorbed antigens facilitate their interaction with immune cell surface receptors, such as Toll-like receptors (TLRs) of antigen-presenting cells.

The chitosan has good stimulation capability of innate immunity and specific immunity, can induce mucosal immunity, has high-efficiency protection property on antigen, and can enhance antigen immunogenicity. The nanoparticles prepared from chitosan can be used as vaccine carriers to accurately load antigens, can be presented to immune cells in a targeted manner, and can be used as an adjuvant to enhance the immunogenicity of the antigens. Compared with the traditional aluminum adjuvant and Freund adjuvant, the chitosan has better antigen protection effect. The antigen can be wrapped in the antigen by the huge molecular weight and the complex spatial structure, and the vaccine is protected by the powerful electrostatic effect due to carrying a large amount of positive charges and is not easy to be enzymolyzed in the transportation process, and the vaccine combined with chitosan has stronger stability than the vaccine directly exposed.

The invention firstly tries the chitosan nano selenium particles as the vaccine carrier, enhances the lymph node delivery of the vaccine and improves the immune effect by adsorbing and wrapping the antigen. The chitosan nano selenium used in the invention has good biological safety, the chitosan nano selenium can be used as a health food or a food additive component to be applied to the food industry, and the chitosan nano selenium particles can obviously improve the immunogenicity of antigens and are suitable for the development of vaccines. Compared with chitosan nano-particles, the chitosan nano-selenium forms nano-selenium particles through selenium reduction reaction without adding an additional cross-linking agent, and has better biological safety. A series of verifications and condition groceries prove that the chitosan nano selenium particles can be used as a carrier or an adjuvant of a protein antigen to generate high immunoreaction aiming at the protein antigen. The strategy provides a novel vaccine carrier, and the immunogenicity of the antigen is effectively improved.

Disclosure of Invention

The first purpose of the invention is to provide chitosan nano selenium particles loaded with antigens and vaccine adjuvants.

The second purpose of the invention is to provide a preparation method of the chitosan nano selenium particles.

The third purpose of the invention is to provide an application of the chitosan nano selenium particle as an antigen carrier or a vaccine adjuvant.

The fourth purpose of the invention is to provide an application of the chitosan nano selenium particles in preparing anti-tumor drugs.

In order to achieve the purpose, the invention provides the following technical scheme:

the first aspect of the invention provides chitosan nano selenium particles loaded with antigens and vaccine adjuvants, wherein positively charged chitosan is subjected to reduction reaction with sodium selenite to form the chitosan nano selenium particles, the chitosan nano selenium particles are spherical, have solid spherical cores and have the particle size range of 20-200 nm.

Further, the PDI of the chitosan nano selenium particles is 0.1-0.4, and the Zeta potential is 15-40 mV.

The second aspect of the present invention provides a method for preparing the chitosan nano selenium particle, comprising the following steps: adding sodium selenite solution into acetic acid solution of chitosan with a certain concentration, magnetically stirring for a certain time at room temperature, adding ascorbic acid solution, reacting for a certain time, standing, aging, dialyzing and purifying through a dialysis bag to obtain the chitosan nano selenium particles.

Further, the concentration ratio of the sodium selenite to the ascorbic acid is 2: 1-1: 20.

Furthermore, the content of the ascorbic acid solution in the reaction liquid is 0-0.50 mol/L.

Further, the concentration of the acetic acid solution of the chitosan is 0.1-5 mg/mL.

Further, the reaction conditions of the standing are as follows: and keeping away from light, wherein the reaction temperature is 0-100 ℃.

The third aspect of the invention provides an application of the chitosan nano selenium particle as an antigen carrier or a vaccine adjuvant, wherein the chitosan nano selenium particle and an antigen with negative electricity form a vaccine particle through an electrostatic adsorption effect or a chemical covalent coupling mode.

Further, the vaccine adjuvant is selected from one of a ligand CpG-ODN of Toll-like receptor 9, a ligand MPLA of Toll-like receptor 4 and an aluminum adjuvant.

Furthermore, the particle size of the vaccine particles is 1-1000 nm, preferably 20-300 nm.

The chitosan nano selenium particles loaded with the antigen and the vaccine adjuvant have the lymph node targeting function, improve the enrichment of the vaccine in the lymph nodes, improve the uptake of the vaccine by antigen presenting cells, improve humoral and cellular immune responses and memory immune responses, and show good effects in the application of cancer prevention.

Therefore, the invention also claims the application of the chitosan nano selenium particles in the preparation of antitumor drugs, the chitosan nano selenium particles and chicken Ovalbumin (OVA) form nanoparticles through electrostatic adsorption, wherein the OVA is used as a model protein for the development and application of antitumor vaccines. After the chitosan nano selenium particles are combined with the OVA mode protein, the chitosan nano selenium particles have stronger immune stimulation capability, the immune stimulation capability is related to the particle size of the chitosan nano selenium particles, and the immune reaction caused by the chitosan nano selenium particles with smaller particle size is stronger.

Further, the particle size of the chitosan nano selenium particles after being combined with OVA protein is 50-300 nm; PdI is 0.1-0.2; the Zeta potential is 15-35 mV.

Compared with the prior art, the invention has the advantages and beneficial effects that:

1. the chitosan nano selenium particles are obtained by the reaction of chitosan and sodium selenite under the reduction of ascorbic acid, no additional cross-linking agent is required to be added, the biological safety is better, and the chitosan nano selenium particles can be used as food additives in the food industry; in addition, the chitosan nano selenium particles can be dissolved in neutral deionized water, and have good physical stability and stability in aqueous solution;

2. the chitosan nano selenium particles can obtain nano particles with different sizes by controlling reaction conditions, can target lymph nodes, and improve the enrichment of antigen substances in the lymph nodes and the uptake of antigen presenting cells, so that the humoral immunity and the cellular immune response of antigen specificity are improved, and the immunological memory capacity is improved;

3. the chitosan nano selenium particles have electropositivity due to the fact that the chitosan has a cationic functional group, can be electrostatically adsorbed with protein antigens with negative electricity, and can be electrostatically adsorbed with the surfaces of cells, so that the chitosan nano selenium particles have better antigen loading capacity and cell targeting capacity; the nano selenium particles can stimulate immune cells to proliferate and can stimulate the production of cytokines, so that the effect of the chitosan nano selenium particles in a vaccine preparation can be improved;

4. the chitosan nano selenium particles of the invention are used as carriers or adjuvants of antigens, show good effects in the application of vaccines, and have great potential to be applied to the vaccine industry.

Drawings

FIG. 1 is a transmission electron microscope image of chitosan nano-selenium particles prepared in example 12;

FIG. 2 is the level of OVA-specific antibodies in mouse serum; (a) mouse serum (diluted 300-fold) ELISA assay at day 28 IgG and IgM mixed antibody levels; (b) group 1, 3 mice sera at 7, 14, 21, 28 days (300 fold dilution) ELISA experiments to measure IgG and IgM mixed antibody levels; (c) group 1, 3 day 28 mouse sera (diluted 300-fold) ELISA assays measured IgG1, IgG2b, IgG3, and IgM antibody levels, respectively; indicates that the third group was significantly different from the first group (P < 0.05).

Detailed Description

The invention is further described with reference to the following figures and examples. The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

The sources of the formulations referred to in the following examples are as follows:

glacial acetic acid and sodium selenite are from national drug group chemical reagent, Inc., L-ascorbic acid and chitosan are from Saen chemical technology (Shanghai) Inc., p-nitrophenyl disodium phosphate hexahydrate, egg white albumin and bovine serum albumin are from Yuanye biological, Inc., and other common reagents are from national drug group chemical reagent, Inc.

Examples 1 to 11

The preparation of the chitosan nano selenium of the invention is realized by changing sodium selenite (Na) in the reaction₂SeO₃) The chitosan nano selenium with different grain diameters is prepared by the proportion of ascorbic acid (Vc) and chitosan, the reaction temperature, the reaction time and other conditions, and the specific preparation method is as follows:

according to the proportion and the reaction conditions shown in the table 1-3, 2.50mL of acetic acid solution of chitosan is put into a 10mL centrifuge tube, 40.00 mu L of sodium selenite solution is added, magnetic stirring is carried out at room temperature for 6h, 200.00 mu L of ascorbic acid solution is added, the acetic acid solution is supplemented to enable the final volume to be 5.00mL, and the mixture is fully mixed and then stands for 12h in a dark place at room temperature or under a heating condition. And finally, filling the chitosan nano selenium solution after the reaction in a dialysis bag with the cut-off molecular weight of 10000kD for dialysis for 24h at room temperature in deionized water, and changing water every 4h to remove unreacted raw materials to obtain the chitosan nano selenium particles of the embodiments 1-11.

TABLE 1 modification of Na₂SeO₃Characterization result of chitosan nano selenium in proportion to Vc concentration

As can be seen from Table 1, the particle size of the chitosan nano-selenium prepared in examples 1-3 increases from 196.0nm to 398.3nm with the content of the reducing agent Vc in the reaction solution, which shows that Na₂SeO₃The concentration ratio of the reducing agent Vc to Vc influences the particle size of the chitosan nano selenium, and in a certain range, the higher the content of the reducing agent Vc in the reaction liquid, the larger the particle size of the prepared chitosan nano selenium is.

TABLE 2 characterization results of chitosan nano-selenium with varying chitosan concentration

As can be seen from Table 2, under the condition that other conditions are not changed, the concentration of the acetic acid solution of the chitosan in the chitosan of examples 4-6 is increased from 1mg/mL to 3mg/mL, and the particle size of the prepared chitosan nano selenium is increased from 103.4nm to 151.8nm, which shows that the concentration of the chitosan is increased and the particle size of the chitosan nano selenium is increased in a certain range.

TABLE 3 characterization results of chitosan nano-selenium with varying reaction temperature and time

As can be seen from Table 3, the particle diameter of the particles showed a tendency to become larger as the temperature of the standing reaction was increased.

Examples 12 to 13

Example 12 is different from example 1, and example 13 is different from example 3 only in that the reaction system is increased from 5mL to 250mL, and the rest of the reaction conditions are not changed, thereby obtaining chitosan nano selenium particles of example 12 and example 13.

As shown in FIG. 1, which is a transmission electron microscope image of the chitosan nano-selenium particle of example 12, it can be seen from FIG. 1 that the chitosan nano-selenium particle of example 12 is spherical, has a solid core inside, and has a particle size of less than 100nm, about 80 nm.

The values of Size, PdI, Zeta and the reaction volume of 5mL measured for the chitosan nanoseles particles of example 12 and example 13 are shown in table 4 below.

TABLE 4 Chitosan nano-selenium prepared with reaction system volumes of 5mL and 250mL

As can be seen from Table 4, the volume of the reaction system has a great influence on the preparation of chitosan nano-selenium, the particle size of the particles is greatly reduced in a 250mL system, the uniformity of the particle size is obviously improved, the Zeta potential absolute value is obviously increased, and the stability of the particles is also obviously improved.

Performance testing

1. Stability test of Chitosan Nanosenium

1.1 physical stability

Taking the chitosan nano selenium particles prepared in the embodiments 2, 3, 4 and 13 as an example, the stability of the chitosan nano selenium particles is tested at normal temperature, and the specific method is as follows:

the chitosan nano-selenium particles prepared in examples 2, 3, 4 and 13 were measured for particle size at day 0 (d0), day 7 (d7), day 30 (d30), and day 60 (d60), respectively, and the results are shown in table 5 below.

TABLE 5 stability of Chitosan Nanosenium at Normal temperature

As can be seen from Table 5, the chitosan nano selenium particles of the four samples have strong stability, no aggregation phenomenon occurs for a long time, the particle size of the sample particles larger than 150nm becomes slightly smaller in a period of time, the particle size of the sample particles smaller than 150nm becomes slightly larger in a period of time, but the overall particle size is more stable with time.

1.2 thermal stability

Taking the chitosan nano selenium particles prepared in the embodiments 12 and 13 as an example, the thermal stability test is carried out, and the specific method is as follows:

the chitosan nano-selenium particles obtained in examples 12 and 13 were placed in a water bath at 50 ℃ for a certain period of time to measure the particle size, and the results are shown in the following table 6:

TABLE 6 thermal stability of Chitosan Nanosenium at 50 deg.C

As can be seen from Table 6, the chitosan nano selenium particles prepared in examples 12 and 13 have good stability at 50 ℃ and small variation degree of particle size.

Example 14

Since the chitosan nano selenium particles prepared in examples 12 and 13 have the highest quality, better particle uniformity and higher Zeta potential, the preparation and characterization of vaccine samples and mouse immunization experiments were carried out using the chitosan nano selenium particles prepared in examples 12 and 13 as samples (respectively labeled as CS-SeNP-12 and CS-SeNP-13).

1. Preparation and characterization of vaccine samples

Preparation of the first vaccine group: OVA + chitosan + aluminium adjuvant (vaccine 1), 2.8mg of chitosan is weighed and dissolved in 0.01mol/L2mL diluted acetic acid solution, then the solution is neutralized by sodium hydroxide solution with equal concentration and equal volume, then the PH is adjusted to 7.4 by using hydrochloric acid with high concentration of 1M, OVA is added, and aluminium adjuvant is added in a ratio of 1:1, and the mixture is uniformly mixed.

Preparation of a second group of vaccines: CS-SeNP-12 (vaccine 2), according to the content of chitosan in the chitosan nano selenium solution, adjusting the concentration of the solution until the content of chitosan is 350 mug/mL.

Preparation of the third group of vaccines: OVA + CS-SeNP-12 (vaccine 3), adding OVA into the chitosan nano selenium solution, and adjusting the concentration of the solution according to the content of chitosan in the chitosan nano selenium solution until the content of chitosan is 350 mug/mL and the content of OVA is 100 mug/mL.

Fourth group vaccine preparation: OVA + CS-SeNP-13 (vaccine 4), as in the third group.

The vaccine characterization results are shown in table 7, and it can be seen from table 7 that the Zeta potential of the particles after OVA addition is reduced, and the Zeta potential value is reduced by about 15%; the particle size is slightly reduced, and the change proportion of the particle size is about 4 percent; all PdI values decreased slightly. Indicating that the particle size tends to be more uniform after OVA is added. These numerical changes can indicate that OVA has adsorbed chitosan nano-selenium surface by electrostatic action.

TABLE 7 vaccine characterization results

2. Immunization experiment of mice

The mouse immunity experiment is divided into four groups, which correspond to the four groups of vaccines respectively, wherein the four groups of vaccines keep the OVA content of 100 mu g/mL and the chitosan content of 350 mu g/mL. Each group of 5 mice was injected subcutaneously with 0.1mL of vaccine per group on days 1, 7, 14, and 21 (i.e., each mouse was injected with 10. mu.g of OVA and 35. mu.g of chitosan). Venous blood of the mice is collected before immunization, at 7 th, 14 th, 21 th and 28 th days respectively, and separated serum is stored in a storage box at the temperature of 80 ℃ below zero and is used for immune activity detection.

3. Vaccine immunocompetence results

After obtaining the mouse antiserum, an enzyme-linked immunosorbent assay (ELISA) is firstly carried out to determine the IgG and IgM mixed antibody titer, the immune effect of the vaccine is preliminarily determined, and the IgG and IgM antibody titer and the antibody titers of different IgG subtypes are further respectively determined. And (3) using OVA as a plating antigen, using goat anti-mouse alkaline phosphatase as a secondary antibody, using a p-nitrophenyl disodium phosphate buffer solution as a color developing agent, developing for 30 minutes at room temperature, measuring absorbance at 415nm of an enzyme-labeling instrument, and representing the level of the OVA specific antibody in the serum of the mouse through the absorbance.

Antibody titer levels are shown in figure 2. As can be seen from fig. 2(a), groups 1, 3 and 4 all produced higher levels of OVA-specific antibody titers at day 28 after 4 immunizations of mice, with group 3 having the highest and significantly higher levels of antibody titers than group 1. From the results, the chitosan nano selenium of the invention has stronger immune stimulation capability, can enhance the immunogenicity of the loaded protein antigen, and the immune reaction stimulated by the chitosan nano selenium loaded OVA antigen with the particle size of about 100nm (group 3 immune results) is obviously higher than the immune level of the group using aluminum agent. The immune effect is slightly reduced along with the increase of the particle size of the chitosan nano selenium. Therefore, the chitosan nano selenium with the length of about 100nm is selected as the vaccine carrier, can effectively stimulate the immunocompetence and is superior to the widely used aluminum agent. As can be seen from FIG. 2(b), the antibody titer in the serum of the mice at days 14, 21 and 28, and the antibody titer caused by the chitosan nano-selenium group of about 100nm, were all higher than the antibody titer caused by the aluminum group, so the chitosan nano-selenium used as the carrier and adjuvant was superior to the existing aluminum group. As can be seen from FIG. 2(c), chitosan nano selenium as the carrier and adjuvant of the vaccine produces antibody types mainly IgG1 and IgG2b, which indicates that the immune response produces better antibody types, and both are higher than the level of the traditional aluminum agent.

In conclusion, the chitosan nano selenium prepared by the invention has stronger immune stimulation capability, can generate stronger immune reaction than the traditional adjuvant aluminum agent, and is expected to become a candidate of an adjuvant or a carrier of a vaccine. The invention discovers that the chitosan nano selenium with the size of 100nm has better effect and can be further used for developing vaccines.

The above description is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto. All equivalent changes, simplifications and modifications which do not depart from the spirit and scope of the invention are intended to be covered by the scope of the invention.