A chalcogenide hybrid silicon nanovaccine adjuvant, its preparation method and application

Abstract

This invention provides a chalcogenide hybrid silicon nanovaccine adjuvant, its preparation method, and its application, belonging to the field of vaccine adjuvant technology. This invention uses bis-[3-(triethoxysilane)propyl]-disulfide and tetraethyl orthosilicate as co-silicon sources, and prepares the chalcogenide hybrid silicon nanovaccine adjuvant through homologous co-doping. By regulating the reaction time of cyclohexane and the co-silicon source to induce the silicon shell topology, a novel biomimetic virus surface nanotopology is constructed on the surface of the vaccine adjuvant. This significantly enhances cellular immune responses, promotes the increase of antigen-specific T lymphocytes in vivo, and generates high-titer neutralizing antibodies in vivo, thereby improving the immunogenicity of the vaccine. Furthermore, in mouse immunization experiments, the vaccine adjuvant not only generates high-titer neutralizing antibodies in humoral immunity but also promotes the uptake of antigen-presenting cells and the activation of downstream immune cells in cellular immunity.

Description

Technical Field

[0001] This invention relates to the field of vaccine adjuvant technology, and in particular to a chalcogenide hybrid silicon nanovaccine adjuvant, its preparation method, and its application. Background Technology

[0002] Adjuvants are auxiliary substances added during vaccine preparation to enhance the immunogenicity of vaccines and improve the level of immune response. The addition of adjuvants can improve antigen stability, increase the efficiency of antigen presentation to immune cells, and promote immune cell activation and antigen processing, thereby enhancing antigen immunogenicity. Due to the immunomodulatory effect of adjuvants, adjuvanted vaccines can often achieve comparable or even better immune effects than traditional vaccines at lower doses. Furthermore, adjuvanted vaccines may require fewer doses to achieve sufficient immune protection. Adjuvants can incorporate multiple identical or different types of antigens into a vaccine to provide broad immune coverage. This allows adjuvanted vaccines to better address pathogen variations, including emerging variants or mutant strains, and even tumors. In addition, adjuvants can be used in combination with different types of vaccine technologies, including traditional inactivated vaccines, recombinant protein vaccines, and emerging nucleic acid vaccines (such as mRNA vaccines) and vector vaccines. This increases the flexibility of vaccine development and selection and helps provide more vaccine options for different diseases.

[0003] Aluminum adjuvants can only induce a Th2-biased immune response, resulting in higher levels of antibody release, but cannot induce Th1-type immune activation. Therefore, aluminum adjuvants cannot produce a strong cellular immune response. Aluminum adjuvants that can only induce humoral immune responses are insufficient to meet the requirements of highly effective vaccines. Summary of the Invention

[0004] The purpose of this invention is to provide a chalcogenide hybrid silicon nanovaccine adjuvant, its preparation method, and its application. The chalcogenide hybrid silicon nanovaccine adjuvant provided by this invention has a virus-mimicking surface nanotopology structure, which can efficiently stimulate humoral and cellular immunity in the body.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing a chalcogenide hybrid silicon nanoparticle vaccine adjuvant, comprising the following steps:

[0007] (1) After mixing tetraethyl orthosilicate, bis-[3-(triethoxysilyl)propyl]-disulfide, ethanol and water, the pH was adjusted to alkaline, and the first heating and stirring and the first post-treatment were carried out in sequence to obtain precursor nanoparticles;

[0008] The precursor nanoparticles and organic solvent were mixed and then subjected to ultrasonic dispersion, centrifugation and deionized water dispersion in sequence to obtain system 1;

[0009] (2) The system 1 obtained in step (1) is mixed with hexadecyltrimethylammonium bromide, water and sodium hydroxide aqueous solution, and then subjected to ultrasonication and second heating and stirring in sequence to obtain system 2;

[0010] (3) The mixture containing cyclohexane, tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide is added dropwise to the system 2 obtained in step (2), and the third heating and stirring and the second post-treatment are carried out in sequence to obtain a solid;

[0011] The solid and an ethanol solution of ammonium nitrate were mixed and subjected to reflux and a third post-treatment to obtain a chalcogenide hybrid silicon nanovaccine adjuvant.

[0012] Preferably, in step (1), the mass ratio of tetraethyl orthosilicate to bis[3-(triethoxysilyl)propyl] disulfide is (3~8):1.

[0013] Preferably, the reagent used to adjust the pH in step (1) is a sodium hydroxide aqueous solution with a concentration of 0.05~1 M; the pH is adjusted to a system pH of 8.5~11.5.

[0014] Preferably, the first heating and stirring time in step (1) is 10~24h, and the temperature of the first heating and stirring is 70~90℃.

[0015] Preferably, in step (2), the mass ratio of hexadecyltrimethylammonium bromide to the precursor nanoparticles in system 1 is (3~9):1.

[0016] Preferably, in step (2), the temperature of the second heating and stirring is 40~90℃, the time of the second heating and stirring is 1~4h, and the speed of the second heating and stirring is 50~300 rpm.

[0017] Preferably, in step (3), tetraethyl orthosilicate accounts for 2 wt% to 12 wt% of system 2, bis[3-(triethoxysilyl)propyl] disulfide accounts for 1 wt% to 5 wt% of system 2, and cyclohexane accounts for 2.5 wt% to 10 wt% of system 2.

[0018] Preferably, the number of reflows in step (3) is 3 to 5, and the time for each reflow is 6 to 48 hours.

[0019] The present invention also provides a chalcogenide hybrid silicon nanovaccine adjuvant prepared by the preparation method described in the above technical solution.

[0020] The present invention also provides a chalcogenide hybrid silicon nanovaccine adjuvant prepared by the preparation method described above, or the application of the chalcogenide hybrid silicon nanovaccine adjuvant in the preparation of nanovaccines.

[0021] This invention provides a method for preparing a chalcogenide hybrid silicon nanovaccine adjuvant. The method involves co-doping a chalcogenide hybrid silicon nanovaccine adjuvant (SS-SiO) using bis-[3-(triethoxysilane)propyl]-disulfide (BTESPD) organosilicon and tetraethyl orthosilicate (TEOS) as co-silicon sources. Then, by controlling the reaction time of cyclohexane and the co-silicon source, a novel biomimetic virus-like surface nanotopological structure is induced on the surface of the adjuvant. Compared with existing technologies, the chalcogenide hybrid silicon nanovaccine adjuvant provided by this invention possesses a biomimetic virus-like surface nanotopological structure, which can significantly enhance cellular immune responses, promote the increase of antigen-specific T lymphocytes in vivo, and generate high-titer neutralizing antibodies in vivo, thereby improving vaccine efficacy. The nanoparticles constructed using the biomimetic virus-like surface topological structure open up a new research direction for vaccine adjuvants. Furthermore, the vaccine adjuvant provided by this invention can generate high titers of neutralizing antibodies in mouse experimental immunization, not only in terms of humoral immunity, but also in terms of cellular immunity, it can promote the uptake of antigen-presenting cells and the activation of downstream immune cells. Attached Figure Description

[0022] Figure 1 The characterization results of the chalcogenide hybrid silicon nanovaccine adjuvant and SNP prepared in Example 1 of this invention are shown in the figure below. Figure 1 As shown, where, Figure 1 The left image of A is a scanning electron microscope image of the SNP. Figure 1 The right image of A in the image is a scanning electron microscope (SEM) image of SS-SiO, with a scale bar of 100 nm. Figure 1 In the figure, B represents the particle size distribution statistics of SNP and SS-SiO. Figure 1 In this context, C represents the zeta potential of SNP and SS-SiO. Figure 1 D represents the statistical analysis of the lengths of the spiky nano-protrusions on the surfaces of SNP and SS-SiO.

[0023] Figure 2 The performance test results of the nanovaccines prepared in Examples 2-4 and Comparative Example 2 of this invention are shown in the figure below. Figure 2 As shown, Figure 2 In the figure, A represents the ratio of SS-SiO bound OVA. Figure 2 In the middle, B represents the OVA protein loading.

[0024] Figure 3 The results of particle size stability and zeta potential stability of the nanovaccine prepared in Example 2 of this invention are shown in the figure below. Figure 3 As shown, where, Figure 3The left and middle figures show particle size stability. Figure 3 The middle right figure shows the stability of the zeta potential;

[0025] Figure 4 The in vitro activation results of BMDCs in the nano-vaccine SS-OVA group, SS-SiO group, OVA group and AL+OVA group of Example 2 of this invention are shown in the figure below. Figure 4 As shown, where, Figure 4 In Figure A, the ability of each vaccine group to promote antigen uptake by dendritic cells (DCs) is shown under a fluorescence microscope. Blue indicates the cell nucleus, red indicates the cell membrane, and green indicates the OVA. The scale bar is 50 μm. Figure 4 In section B, flow cytometry was used to detect the ability of each vaccine group to induce the maturation and differentiation of BMDCs. Figure 4 In the middle C, the level of TNF-α secreted by BMDCs stimulated by each vaccine group is represented.

[0026] Figure 5 The following is a graph showing the results of OVA antigen-specific serum antibody production in mice induced by the nano-vaccines in Example 2 of this invention: SS-OVA group, SS-SiO group, OVA group, and AL+OVA group. Figure 5 As shown, where, Figure 5 In the middle section, A represents the titer of OVA-specific total IgG antibodies in the serum of mice within ten weeks after the initial immunization. Figure 5 In the middle B, the ratio of IgG1 antibody titer to IgG2 antibody titer in the serum of mice six weeks after the initial immunization is given.

[0027] Figure 6 The following figures show the in vivo cellular immune response results induced by the nano-vaccines in Example 2 of this invention, specifically in the SS-OVA group, SS-SiO group, OVA group, AL+OVA group, and Control group. Figure 6 As shown, where, Figure 6 In the middle, A represents the content of T lymphocytes. Figure 6 In the middle B, the Th content of T helper lymphocytes is specific to OVA. Figure 6 In the middle, C represents the content of memory cells in T cell cytotoxic lymphocytes (Tc). Figure 6 D represents the content of memory cells among T helper lymphocytes (Th).

[0028] Figure 7 The images show the H&E staining results of tissue sections from the heart, liver, spleen, lung, and kidney of mice in the Control and SS-OVA groups in this invention. Detailed Implementation

[0029] This invention provides a method for preparing a chalcogenide hybrid silicon nanoparticle vaccine adjuvant, comprising the following steps:

[0030] (1) After mixing tetraethyl orthosilicate, bis-[3-(triethoxysilyl)propyl]-disulfide, ethanol and water, the pH was adjusted to alkaline, and the first heating and stirring and the first post-treatment were carried out in sequence to obtain precursor nanoparticles;

[0031] The precursor nanoparticles and organic solvent were mixed and then subjected to ultrasonic dispersion, centrifugation and deionized water dispersion in sequence to obtain system 1;

[0032] (2) The system 1 obtained in step (1) is mixed with hexadecyltrimethylammonium bromide, water and sodium hydroxide aqueous solution, and then subjected to ultrasonication and second heating and stirring in sequence to obtain system 2;

[0033] (3) The mixture containing cyclohexane, tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide is added dropwise to the system 2 obtained in step (2), and the third heating and stirring and the second post-treatment are carried out in sequence to obtain a solid;

[0034] The solid and an ethanol solution of ammonium nitrate were mixed and subjected to reflux and a third post-treatment to obtain a chalcogenide hybrid silicon nanovaccine adjuvant.

[0035] Unless otherwise specified, all raw materials used in this invention are commercially available products in the art.

[0036] In this invention, tetraethyl orthosilicate, bis-[3-(triethoxysilyl)propyl]-disulfide, ethanol and water are mixed, the pH is adjusted to alkaline, and then subjected to a first heating and stirring and a first post-treatment to obtain precursor nanoparticles.

[0037] In this invention, the method for preparing the bis[3-(triethoxysilyl)propyl]-disulfide preferably includes the following steps: adding γ-chloropropyltriethoxysilane dropwise to a sodium disulfide solution, and sequentially stirring, stopping the reaction, extracting, drying, and chromatographic purification to obtain the bis[3-(triethoxysilyl)propyl]-disulfide.

[0038] In this invention, the molar ratio of γ-chloropropyltriethoxysilane to sodium disulfide in the sodium disulfide solution is preferably (1.5~3):1, more preferably 2:1. In this invention, the stirring time is preferably 8~12 h, more preferably 10 h. In this invention, the method for stopping the reaction is preferably adding an ice-water mixture. In this invention, the organic solvent used for extraction is preferably dichloromethane. In this invention, the drying method is preferably drying the organic layer obtained from the extraction with anhydrous sodium sulfate. In this invention, the chromatographic purification method is preferably silica gel column chromatography; the solvent used for silica gel column chromatography is preferably a mixed solvent composed of petroleum ether (PE) and dichloromethane (DCM) in a volume ratio of (10~1):1.

[0039] In this invention, the mass ratio of tetraethyl orthosilicate to bis[3-(triethoxysilyl)propyl] disulfide is preferably (3~8):1, more preferably (4~7):1, and even more preferably 5:1. This invention controls the mass ratio of tetraethyl orthosilicate to bis[3-(triethoxysilyl)propyl] disulfide within the above range to control the amount of chalcogenide doping, thereby regulating the degradation efficiency of nanoparticles under acidic conditions. In this invention, the ethanol is preferably anhydrous ethanol; the amount of anhydrous ethanol used is 2.5~10 wt% of the mixture of tetraethyl orthosilicate, bis[3-(triethoxysilyl)propyl]-disulfide, ethanol, and water, more preferably 5 wt%. In this invention, the reagent used to adjust the pH is preferably a 0.05~1 M sodium hydroxide aqueous solution, more preferably a 0.1 M sodium hydroxide aqueous solution; the pH of the system is preferably adjusted to 8.5~11.5, more preferably 9.5. This invention controls the reaction rate by adjusting the pH, which facilitates the regulation of the precursor nanoparticle size. In this invention, the first heating and stirring time is preferably 10-24 hours, more preferably 12 hours; the first heating and stirring temperature is preferably 70-90°C, more preferably 75-85°C. Controlling the first heating and stirring time and temperature within the above ranges helps to control and homogenize the precursor nanoparticle size. In this invention, the first post-treatment preferably includes sequentially centrifuging, washing with anhydrous ethanol, and vacuum drying of the product from the first heating and stirring. In this invention, the number of anhydrous ethanol washes is preferably 3-5. This invention does not have specific limitations on the vacuum drying method, achieving the removal of residual solvents.

[0040] After obtaining the precursor nanoparticles, the present invention mixes the precursor nanoparticles with an organic solvent and then performs ultrasonic dispersion, centrifugation and deionized water dispersion in sequence to obtain system 1.

[0041] In this invention, the organic solvent is preferably anhydrous ethanol. In this invention, the ultrasonic dispersion time is preferably 12-20 min. In this invention, the concentration of the precursor nanoparticles in system 1 is preferably 2-10 g / L, more preferably 2-10 g / L, and even more preferably 5 g / L.

[0042] After obtaining system 1, the present invention mixes system 1 with hexadecyltrimethylammonium bromide, water and sodium hydroxide aqueous solution, and then performs ultrasonication and second heating and stirring in sequence to obtain system 2.

[0043] In this invention, the preferred mass ratio of the hexadecyltrimethylammonium bromide to the precursor nanoparticles in system 1 is (3-9):1, more preferably (4-8):1, and even more preferably 5:1. This invention controls the mass ratio of the hexadecyltrimethylammonium bromide to the precursor nanoparticles in system 1 within the above range to control the thickness of the nanoparticle surface shell, thereby regulating the surface morphology of the nanoparticles. In this invention, the preferred concentration of the sodium hydroxide solution is 0.04-0.2 mM, more preferably 0.08-0.15 mM; the preferred volume ratio of system 1 to the sodium hydroxide solution is 200:3. This invention controls the amount of sodium hydroxide solution within the above range to control the reaction rate, which is beneficial for regulating the surface morphology of the nanoparticles. In this invention, the preferred ultrasonication time is 5-20 min, more preferably 8-15 min; the preferred ultrasonication power is 200-500 W, more preferably 300 W. This invention uses ultrasonication to fully disperse the nanoparticles, which is beneficial for subsequent control of nanoparticle morphology and homogenization. In this invention, the temperature of the second heating and stirring is preferably 40-90℃, more preferably 50-85℃, and even more preferably 60℃; the time of the second heating and stirring is preferably 1-4h, more preferably 1.5-3.5h, and even more preferably 2h; the rotation speed of the second heating and stirring is preferably 50-300 rpm, more preferably 80-260 rpm, and even more preferably 100 rpm. This invention controls the temperature, time, and rotation speed of the second heating and stirring within the above ranges to control the morphology and homogenization of the nanoparticles.

[0044] After obtaining system 2, the present invention adds dropwise a mixture containing cyclohexane, tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide to system 2, and performs a third heating and stirring and a second post-treatment in sequence to obtain a solid.

[0045] In this invention, the tetraethyl orthosilicate preferably accounts for 2 wt% to 12 wt% of the system 2, more preferably 2 wt% to 12 wt%, and even more preferably 10 wt%. In this invention, the bis[3-(triethoxysilyl)propyl] disulfide accounts for 1 wt% to 5 wt% of the system 2, more preferably 2 wt% to 4 wt%, and even more preferably 2.5 wt%. In this invention, the cyclohexane accounts for 2.5 wt% to 10 wt% of the system 2, more preferably 3 wt% to 8 wt%, and even more preferably 5 wt%. This invention controls the amounts of tetraethyl orthosilicate, bis[3-(triethoxysilyl)propyl] disulfide, and cyclohexane within the above ranges to control the amount of chalcogenide doping, thereby regulating the degradation efficiency of nanoparticles under acidic conditions. In this invention, the third heating and stirring time is preferably 24-96 hours, more preferably 48-84 hours, and even more preferably 72 hours; the third heating and stirring temperature is preferably 40-90°C, more preferably 50-85°C, and even more preferably 60°C. This invention controls the temperature and time of the third heating and stirring within the above ranges to obtain nanoparticles with medium-length surface spike structures, forming a biomimetic viral surface nanotopology, improving the cellular immune response of the subsequently prepared chalcogenide hybrid silicon nanovaccine adjuvant, thereby promoting the increase of antigen-specific T lymphocytes in vivo and generating high-titer neutralizing antibodies, thus improving the immunogenicity of the vaccine.

[0046] In this invention, the second post-processing includes sequentially centrifuging, washing with ethanol, and washing with deionized water to obtain a solid. Preferably, the number of ethanol washes and the number of deionized water washes are 3 to 5.

[0047] After obtaining the solid, the present invention mixes the solid with an ethanol solution of ammonium nitrate, and performs reflux and a third post-treatment in sequence to obtain a chalcogenide hybrid silicon nanovaccine adjuvant.

[0048] In this invention, the concentration of ammonium nitrate in the ammonium nitrate ethanol solution is preferably 0.5 wt% to 2 wt%.

[0049] In this invention, the reflux is preferably performed 3 to 5 times; the duration of each reflux is preferably 6 to 48 hours, more preferably 8 to 32 hours, and even more preferably 12 hours. In this invention, the reflux is preferably performed under stirring conditions; after each reflux, the process preferably further includes: centrifuging the refluxed product sequentially, washing with ethanol, and then performing the next reflux. In this invention, the third post-treatment preferably includes centrifuging the refluxed product sequentially, washing with ethanol, and then vacuum drying.

[0050] The present invention does not impose any special limitations on the vacuum drying method; conventional technical solutions in the field can be used.

[0051] The present invention also provides a chalcogenide hybrid silicon nanovaccine adjuvant prepared by the preparation method described in the above technical solution.

[0052] The present invention also provides a chalcogenide hybrid silicon nanovaccine adjuvant prepared by the preparation method described above, or the application of the chalcogenide hybrid silicon nanovaccine adjuvant in the preparation of nanovaccines.

[0053] In this invention, the nano-vaccine is preferably at least one of OVA model vaccines, cancer vaccines, COVID-19 vaccines, and swine fever vaccines.

[0054] In this invention, the method for preparing a nanovaccine using the chalcogenide hybrid silicon nanovaccine adjuvant prepared by the above technical solution preferably includes the following steps:

[0055] After mixing the chalcogenide hybrid silicon nanovaccine adjuvant with the vaccine antigen, the mixture was sequentially shaken, centrifuged, and dispersed in a solvent to obtain the nanovaccine.

[0056] In this invention, the antigen is preferably at least one selected from OVA chicken ovalbumin, melanoma B16F10 cell membrane protein, RBD, and E2 subprotein. In this invention, the oscillation time is preferably 9-18 hours, more preferably 10-16 hours, and even more preferably 12 hours. In this invention, the oscillation temperature is preferably 4°C. In this invention, the mass ratio of the vaccine antigen to the chalcogenide hybrid silicon nanovaccine adjuvant is preferably 1:(1-10), more preferably 1:(2-8), and even more preferably 1:5. This invention activates APC through multiple signaling pathways, promoting APC antigen presentation efficiency, and obtaining a nanovaccine that can efficiently stimulate humoral and cellular immunity.

[0057] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0058] Unless otherwise specified, all experiments were repeated three times, and the results are expressed as averages.

[0059] Example 1

[0060] A method for preparing a chalcogenide hybrid silicon nanoparticle vaccine adjuvant, comprising the following steps:

[0061] (1) The preparation method of bis[3-(triethoxysilyl)propyl] disulfide, the steps are as follows:

[0062] 10 g of γ-chloropropyltriethoxysilane was added dropwise to 30 mL of sodium disulfide solution (containing 0.0255 mol of sodium disulfide). The mixture was stirred overnight (10 h) at room temperature (25 °C). The reaction was stopped by adding an ice-water mixture. The mixture was extracted with dichloromethane. The organic layer was dried with anhydrous sodium sulfate and then purified by silica gel column chromatography (300-400 mesh) (the volume ratio of petroleum ether (PE): dichloromethane (DCM) was 10-1:1). The resulting milky white liquid was bis[3-(triethoxysilyl)propyl]disulfide.

[0063] The molar ratio of the γ-chloropropyltriethoxysilane to the sodium disulfide in the sodium disulfide solution is 2:1;

[0064] Take 5 g of tetraethyl orthosilicate, 1 g of the above-prepared bis[3-(triethoxysilyl)propyl]disulfide and 2 mL of anhydrous ethanol into a round-bottom flask, add 40 mL of ultrapure water to dissolve them completely, adjust the pH of the system to 9.5 by adding 0.1 M sodium hydroxide dropwise, heat to 80 °C for the first heating and stirring for 12 h, then collect the nanoparticles by centrifugation, wash them thoroughly three times with anhydrous ethanol, and finally dry them under vacuum to obtain the precursor nanoparticles (abbreviated as SNP).

[0065] The mass ratio of tetraethyl orthosilicate to bis[3-(triethoxysilyl)propyl] disulfide is 5:1; the amount of anhydrous ethanol used is 5 wt% of the mixture of tetraethyl orthosilicate, bis[3-(triethoxysilyl)propyl] disulfide, ethanol and water.

[0066] 100 mg of the precursor nanoparticles were dispersed in anhydrous ethanol and sonicated for 15 min to ensure uniform dispersion. The particles were then collected by centrifugation and redispersed in 20 mL of deionized water to obtain system 1 with a preferred concentration of 5 g / L of precursor nanoparticles.

[0067] (2) Add 30 mL of deionized water, 0.5 g of hexadecyltrimethylammonium bromide and 0.3 mL of sodium hydroxide (0.1 mM) to the flask. After complete dissolution, a mixture is obtained.

[0068] Add system 1 obtained in step (1) to the mixture and sonicate at 300W for 10 min. Then, heat and stir for 2 h at 100 rpm / min in an oil bath at 60°C to obtain system 2.

[0069] The mass ratio of the hexadecyltrimethylammonium bromide to the precursor nanoparticles in system 1 is 5:1;

[0070] (3) The mixture containing 16 mL cyclohexane, 4 mg tetraethyl orthosilicate and 1 mg bis[3-(triethoxysilyl)propyl] disulfide was added dropwise to system 2 obtained in step (2). The mixture was heated and stirred at 60 °C for 72 h. The solid was collected by centrifugation, washed with ethanol and deionized water. The solid and an ethanol solution of 1 wt% ammonium nitrate were refluxed for 12 h, collected by centrifugation, washed with ethanol 3 times, and then refluxed twice. Finally, the solid was collected by centrifugation, washed with ethanol 3 times and vacuum dried to obtain the chalcogen hybrid silicon nanovaccine adjuvant SS-SiO.

[0071] The tetraethyl orthosilicate preferably accounts for 10 wt% of system 2; the bis[3-(triethoxysilyl)propyl] disulfide accounts for 2.5 wt% of system 2; and the cyclohexane accounts for 5 wt% of system 2.

[0072] Comparative Example 1

[0073] The preparation method of SNPs includes the following steps:

[0074] The preparation method of bis[3-(triethoxysilyl)propyl] disulfide includes the following steps:

[0075] 10 g of γ-chloropropyltriethoxysilane was added dropwise to 30 mL of sodium disulfide solution (containing 0.0255 mol of sodium disulfide). The mixture was stirred overnight (10 h) at room temperature (25 °C). The reaction was stopped by adding an ice-water mixture. The mixture was extracted with dichloromethane. The organic layer was dried with anhydrous sodium sulfate and then purified by silica gel column chromatography (300-400 mesh) (the volume ratio of petroleum ether (PE): dichloromethane (DCM) was 10-1:1). The resulting milky white liquid was bis[3-(triethoxysilyl)propyl]disulfide.

[0076] The molar ratio of the γ-chloropropyltriethoxysilane to the sodium disulfide in the sodium disulfide solution is 2:1;

[0077] Take 5 g of tetraethyl orthosilicate, 1 g of the above-prepared bis[3-(triethoxysilyl)propyl]disulfide and 2 mL of anhydrous ethanol into a round-bottom flask, add 40 mL of ultrapure water to dissolve them completely, adjust the pH of the system to 9.5 by adding 0.1 M sodium hydroxide dropwise, heat to 80 °C for the first heating and stirring for 12 h, then collect the nanoparticles by centrifugation, wash them thoroughly three times with anhydrous ethanol, and finally dry them under vacuum to obtain the precursor nanoparticles (abbreviated as SNP).

[0078] The mass ratio of tetraethyl orthosilicate to bis[3-(triethoxysilyl)propyl] disulfide is 5:1; the amount of anhydrous ethanol used is 5 wt% of the mixture of tetraethyl orthosilicate, bis[3-(triethoxysilyl)propyl] disulfide, ethanol and water.

[0079] The products of Example 1 and Comparative Example 1 were observed using scanning electron microscopy (SEM), and their particle size distribution and zeta potential were detected using dynamic light scattering. The length of the surface spiked nanoparticles was also observed using SEM. The characterization results of the chalcogenide hybrid silicon nanovaccine adjuvant and SNP prepared in Example 1 are shown in the figure below. Figure 1 As shown, where, Figure 1 The left image of A is a scanning electron microscope image of the SNP. Figure 1 The right image of A in the image is a scanning electron microscope (SEM) image of SS-SiO, with a scale bar of 100 nm. Figure 1 In the figure, B represents the particle size distribution statistics of SNP and SS-SiO. Figure 1 In this context, C represents the zeta potential of SNP and SS-SiO. Figure 1 D represents the statistical length of the spiked nanospikes on the surface of SNPs and SS-SiO. Figure 1 It can be seen that the chalcogenide hybrid silicon nanovaccine adjuvant SS-SiO prepared in Example 1 has a biomimetic virus surface nanotopological structure, uniform particle size, particle diameter of about 150 nm, zeta potential of about -27 mV, surface spiky structure length of about 15 nm and diameter of about 9 nm, while the SNP surface is smooth.

[0080] Example 2

[0081] The application of chalcogenide hybrid silicon nanovaccine adjuvants in the preparation of nanovaccines, the steps are as follows:

[0082] OVA chicken egg albumin was mixed with the chalcogenide hybrid silicon nanovaccine adjuvant SS-SiO prepared in Example 1 at a mass ratio of 1:5. The mixture was shaken and mixed overnight at 4°C for 12 h. The nanovaccine was collected by centrifugation (also known as a nanovaccine based on chalcogenide hybrid silicon nanovaccine adjuvant). The prepared nanovaccine was redispersed in PBS and sonicated at 4°C for 15 min to obtain the SS-OVA group (1:5) of nanovaccines that can efficiently stimulate humoral and cellular immunity in the body. The final concentration was 1 mg / mL (calculated based on the total protein content of OVA).

[0083] Example 3

[0084] The nanovaccine SS-OVA group (1:1) was prepared according to the method of Example 2. The difference from Example 1 is that the OVA chicken egg albumin was prepared in a mass ratio of 1:1 to the sulfhydryl hybrid silicon nanovaccine adjuvant SS-SiO prepared in Example 1.

[0085] Example 4

[0086] The nanovaccine SS-OVA group (1:10) was prepared according to the method of Example 2. The difference from Example 1 is that the OVA chicken egg albumin was prepared in a mass ratio of 1:10 to the chalcogen hybrid silicon nanovaccine adjuvant SS-SiO prepared in Example 1.

[0087] Comparative Example 2

[0088] The nanovaccine SS-OVA group (1:20) was prepared according to the method of Example 2. The difference from Example 1 is that the OVA chicken egg albumin was prepared in a mass ratio of 1:20 to the chalcogen-hybrid silicon nanovaccine adjuvant SS-SiO prepared in Example 1.

[0089] Comparative Example 3

[0090] The SS-SiO prepared in Example 1 was dispersed in PBS and sonicated at 4°C for 15 min to obtain a free SS-SiO group with a concentration of 200 μg / mL.

[0091] OVA was dispersed in PBS and sonicated at 4°C for 15 min to obtain a free antigen OVA group with a concentration of 1 mg / mL.

[0092] The aluminum adjuvant and OVA were mixed and sonicated at 4°C for 15 min to obtain the AL+OVA group; the aluminum adjuvant was Bioss C5085 aluminum hydroxide adjuvant (alum adjuvant).

[0093] The ratio of SS-SiO binding to OVA in the nanovaccines prepared in Examples 2-4 and Comparative Example 2 was detected by flow cytometry. The OVA protein loading in the nanovaccines prepared in Examples 2-4 and Comparative Example 2 was quantitatively detected by BCA. The performance test results of the nanovaccines prepared in Examples 2-4 and Comparative Example 2 are shown in the figure below. Figure 2 As shown, Figure 2 In the figure, A represents the ratio of SS-SiO bound OVA. Figure 2 In the figure, B represents the OVA protein loading. (From...) Figure 2 It can be seen that the efficiency of adjuvant nanoparticle binding to FITC-OVA detected by flow cytometry is close to 100%. Quantitative detection by BCA can infer that 500 μg of the chalcogen-hybrid silicon nanoparticle adjuvant nanoparticles encapsulates approximately 50 μg of OVA protein.

[0094] The particle size stability and zeta potential stability of the nanovaccine prepared in Example 2 were detected using dynamic light scattering (DLS). The results are shown in the figure below. Figure 3 As shown, where, Figure 3 The left and middle figures show particle size stability. Figure 3 The right-hand figure shows the stability of the zeta potential. (From...) Figure 3 It can be seen that the nano-vaccine prepared in Example 2 has good dispersibility and stability in water, and exhibits good stability within 7 days.

[0095] Investigation 1: Activation of BMDC in vitro by the nanovaccine prepared in Example 2

[0096] The nanovaccine SS-OVA group from Example 2 was added to cultured BMDC cells at a concentration of 1 μg / mL. The SS-SiO group, OVA group, and AL+OVA group were treated in the same way. BMDCs treated with the same volume of PBS served as the control group. After incubation for 4 h, the ability of BMDC cells to take up the nanovaccine was detected by fluorescence microscopy and flow cytometry. After incubation for 24 h, BMDC cells were collected, and the maturation and differentiation ability of BMDCs stimulated by the nanovaccine was detected by flow cytometry using the DC maturation markers CD80 and CD86. The cell culture supernatant was collected, and the TNF-α level was detected by ELISA.

[0097] The in vitro activation results of BMDCs in the nano-vaccine groups SS-OVA, SS-SiO, OVA, and AL+OVA of Example 2 are shown in the figure below. Figure 4 As shown, where, Figure 4 In Figure A, the ability of each vaccine group to promote antigen uptake by dendritic cells (DCs) is shown under a fluorescence microscope. Blue indicates the cell nucleus, red indicates the cell membrane, and green indicates the OVA. The scale bar is 50 μm. Figure 4 In section B, flow cytometry was used to detect the ability of each vaccine group to induce the maturation and differentiation of BMDCs. Figure 4 In the middle, C represents the level of TNF-α secreted by BMDCs stimulated by each vaccine group. Figure 4 The results showed that, under a fluorescence microscope, the co-localization fluorescence of OVA-FITC and the cell membrane revealed that the SS-OVA group exhibited the best antigen uptake capacity in DCs. Flow cytometry analysis indicated that SS-OVA had a significantly superior ability to stimulate BMDC maturation and differentiation compared to other groups. Furthermore, the nanovaccine based on chalcogenide hybrid nanosilicon adjuvant, which can efficiently stimulate humoral and cellular immunity, can stimulate BMDC cells to release the cytoinflammatory cytokine TNF-α, suggesting its potential contribution to immune activation in in vivo experiments.

[0098] Investigation 2: The effect of the nanovaccine prepared in Example 2 on in vivo in inducing humoral immune response.

[0099] The nano-vaccines from Example 2, namely the SS-OVA group, SS-SiO group, OVA group, and AL+OVA group, were administered subcutaneously at a dose of 200 μL to immunize 5-week-old female BALB / c mice, with immunizations occurring twice, once at week 0 and once at week 2. Blood samples were collected every two weeks, and the serum titers of OVA-specific total IgG, IgG1, and IgG2 antibodies were detected by ELISA.

[0100] The results of detecting the OVA antigen-specific serum antibodies produced in mice induced by the nano-vaccines of Example 2 (SS-OVA group, SS-SiO group, OVA group, and AL+OVA group) are shown in the figure below. Figure 5 As shown, where, Figure 5 In the middle section, A represents the titer of OVA-specific total IgG antibodies in the serum of mice within ten weeks after the initial immunization. Figure 5 In the middle B, the ratio of IgG1 antibody titer to IgG2 antibody titer in the serum of mice six weeks after initial immunization is given. Figure 5 It was found that from the second to the fourth week after the initial immunization, the serum of mice in the SS-OVA group showed a significant increase in OVA-specific total IgG antibodies, peaking at the sixth week. Compared with other vaccines, SS-OVA immunization resulted in the best serum antibody titer, and the serum antibody titer remained at a relatively high level (approximately 10) from the sixth to the tenth week thereafter. 5 This demonstrates the good humoral immune effect of the vaccine, confirming the effectiveness of SS-OVA. Furthermore, at week six, the IgG1 and IgG2 antibody titers in the SS-OVA group mice were less than 1, indicating that SS-OVA induces a Th1-oriented immune effect, while the aluminum adjuvant induces a Th2-oriented immune effect, suggesting the potential of SS-OVA to efficiently stimulate both humoral and cellular immunity.

[0101] Investigation 3: The effect of the nano-vaccine prepared in Example 2 on in vivo cellular immune response.

[0102] In the second experimental model, at week four after the initial immunization, spleen cells from mice were extracted and analyzed by flow cytometry to detect changes in the content of immune cells, including T-cell cytotoxic lymphocytes (Tc) and T-helper lymphocytes (Th), as well as corresponding memory and effector cells. Age-matched unimmunized mice served as the control group.

[0103] The results of detecting the in vivo cellular immune effects of the nano-vaccines in Example 2 in mice, including the SS-OVA group, SS-SiO group, OVA group, AL+OVA group, and Control group, are shown in the figure below. Figure 6 As shown, where, Figure 6 In the middle, A represents the content of T lymphocytes. Figure 6 In the middle B, the Th content of T helper lymphocytes is specific to OVA. Figure 6In the middle, C represents the content of memory cells in T cell cytotoxic lymphocytes (Tc). Figure 6 D represents the content of memory cells among T helper lymphocytes (Th cells). (From...) Figure 6 It can be seen that, Figure 6 Figure A shows that after mice were immunized with the SS-OVA vaccine, the number of T lymphocytes was significantly higher than that in other groups, indicating that SS-OVA effectively stimulated the cellular immune process. Figure 6 The content of OVA-specific T helper lymphocytes (Th cells) in mice also showed a significant increase, indicating that cellular immunity in mice was induced by the OVA vaccine. Figure 6 In groups C and D, the content of memory cells in T-cell cytotoxic lymphocytes (Tc) and T-helper lymphocytes (Th) was also significantly higher than in other groups, indicating that if the mouse's immune system is attacked again, it can quickly initiate a response and produce a cellular immune effect.

[0104] Investigation 3: In vivo tissue safety of the nanovaccine prepared in Example 2

[0105] In the second experimental model, at week 10 post-primary immunization, heart, liver, spleen, lung, and kidney tissues were collected from age-matched unexperimented mice (Control group) and SS-OVA group mice. Tissue slides were prepared, and HE staining was used to assess the tissue safety of the nanovaccine in vivo. Age-matched unimmunized mice served as the control group.

[0106] The H&E staining results of tissue sections from the heart, liver, spleen, lung, and kidney of mice in the Control and SS-OVA groups are shown in the figure below. Figure 7 As shown, by Figure 7 It was found that, 10 weeks after immunization, no obvious tissue toxicity was observed in the tissue sections of the SS-OVA group mice, indicating that the nanovaccine based on chalcogenide hybrid silicon nanovaccine adjuvant prepared in Example 2 has good tissue safety in vivo.

[0107] In summary, the chalcogenide hybrid silicon nanovaccine adjuvant prepared by the method provided by this invention has a biomimetic virus surface nanotopological structure. In mouse experimental immunization, it can not only produce high titers of neutralizing antibodies in humoral immunity, but also promote the uptake of antigen-presenting cells and the activation of downstream immune cells in cellular immunity.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a chalcogen-hybridized silicon nano-adjuvant for vaccines, comprising the following steps: (1) mixing tetraethyl orthosilicate, bis-[3-(triethoxysilyl)propyl]-disulfide, ethanol and water, adjusting the pH to alkaline, sequentially performing first heating stirring and first post-treatment to obtain precursor nanoparticles; mixing the precursor nanoparticles and an organic solvent, sequentially performing ultrasonic dispersion, centrifugation and deionized water dispersion to obtain system 1; (2) mixing system 1 obtained in step (1) with cetyltrimethylammonium bromide, water and sodium hydroxide aqueous solution, sequentially performing ultrasonic and second heating stirring to obtain system 2; (3) adding a mixture containing cyclohexane, tetraethyl orthosilicate and bis-[3-(triethoxysilyl)propyl]-disulfide dropwise into system 2 obtained in step (2), sequentially performing third heating stirring and second post-treatment to obtain a solid; mixing the solid and an ethanol solution of ammonium nitrate, sequentially performing reflux and third post-treatment to obtain the chalcogen-hybridized silicon nano-adjuvant for vaccines.

2. The production method according to claim 1, characterized by, In step (1), the mass ratio of tetraethyl orthosilicate to bis-[3-(triethoxysilyl)propyl]-disulfide is (3-8):

1.

3. The preparation method according to claim 1, characterized in that, In step (1), the reagent used for adjusting the pH is a sodium hydroxide aqueous solution with a concentration of 0.05-1 M; and the pH of the system after adjusting the pH is 8.5-11.

5.

4. The method of claim 1, wherein, In step (1), the first heating stirring is performed for 10-24 h at a temperature of 70-90℃.

5. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of cetyltrimethylammonium bromide to the precursor nanoparticles in system 1 is (3-9):

1.

6. The method of claim 1, wherein, In step (2), the second heating stirring is performed at a temperature of 40-90℃ for 1-4 h at a rotation speed of 50-300 rpm.

7. The preparation method according to claim 1, characterized in that, In step (3), the content of tetraethyl orthosilicate in system 2 is 2 wt%-12 wt%, the content of bis-[3-(triethoxysilyl)propyl]-disulfide in system 2 is 1 wt%-5 wt%, and the content of cyclohexane in system 2 is 2.5 wt%-10 wt%.

8. The method of claim 1, wherein, In step (3), the reflux is performed for 3-5 times, and each reflux is performed for 6-48 h. 9.A chalcogen-hybridized silicon nano-adjuvant for vaccines prepared by the method of any one of claims 1-8. 10.Use of the chalcogen-hybridized silicon nano-adjuvant for vaccines of claim 9 in preparing a nano-vaccine.

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