CN113896206A - Hollow mesoporous silicon nano-microsphere and preparation method and application thereof - Google Patents
Hollow mesoporous silicon nano-microsphere and preparation method and application thereof Download PDFInfo
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- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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Abstract
The invention belongs to the technical field of materials, and particularly relates to a hollow mesoporous silicon nano microsphere as well as a preparation method and application thereof. The nano-microspheres are spherical; the interior of the shell is a cavity structure, the size of the cavity is 150 +/-10 nm, the exterior of the shell is a mesoporous silicon shell layer, and the thickness of the shell layer is 55 +/-5 nm. The shell layer of the nano microsphere is provided with a mesoporous structure of 3 +/-1 nm. The hollow mesoporous silicon nano microsphere has the advantages of good safety, high stability, simple preparation and the like, and can be used for preparing foot-and-mouth disease virus-like particle vaccines.
Description
Technical Field
The invention belongs to the technical field of materials, and particularly relates to a hollow mesoporous silicon nano microsphere as well as a preparation method and application thereof.
Background
Foot-and-mouth disease is an acute, febrile and highly contagious disease caused by foot-and-mouth disease virus (FMDV) and mainly caused by important economic livestock such as cattle, pigs, sheep and the like. Vaccination is one of the most effective means for the prevention and control of foot and mouth disease at present. Although the inactivated vaccine of foot-and-mouth disease plays an extremely important role in the effective prevention and control of foot-and-mouth disease, the inactivated vaccine needs to use live virus in the production process, the live virus is not inactivated completely or the production process is not treated properly, so that the danger of causing epidemic situation exists.
In recent years, with the vigorous development of molecular biology, molecular immunology, reverse genetics, bioinformatics and other disciplines, a large number of novel vaccines such as live vector vaccines, synthetic peptide vaccines, virus-like particle vaccines and the like have been widely studied. Among them, virus-like particle vaccines are considered as the best candidate vaccine form that can replace traditional inactivated virus vaccines. Although the virus-like particle vaccine has better safety compared with the inactivated vaccine, the problems of weak immunogenicity, poor stability and stress resistance and the like become the bottleneck of the development of the vaccine at present.
The adjuvant is also called as an immunomodulator or an immunopotentiator, and has the functions of reducing the number of immunization injections, reducing the dosage of antigens, enhancing the immune response and the like. Conventional adjuvants include insoluble aluminum salt colloids, oil-water emulsions, microorganisms and their metabolites, nucleic acids and their analogs, cytokines, immunostimulatory complexes, propolis, liposomes, and the like.
At present, the adjuvant used by the foot-and-mouth disease vaccine mainly uses ISA series oil-water emulsion. The adjuvant is imported for a long time, is influenced by factors such as patent protection and the like, and is expensive, but few commercial adjuvants which are independently researched and developed at home at present. Therefore, the research and development of a novel vaccine adjuvant which is efficient, safe, stable and economical is urgently needed, and important contributions are made for breaking international monopoly, exploring the potential of domestic vaccine enterprises and developing novel genetic engineering subunit vaccines.
In recent years, mesoporous silicon nanomaterials have been attracting more and more attention as carriers of vaccine delivery systems due to their characteristics of low toxicity, easy degradation, easy surface functionalization modification, and the like. The search for safe, stable and effective novel nano-adjuvant for the original granular vaccine of the foot-and-mouth disease has important significance.
Disclosure of Invention
The invention aims to provide a hollow mesoporous silicon nano microsphere with large specific surface area and pore volume, uniform particle size, high biological safety, high stability and simple preparation, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a hollow mesoporous silicon nanometer microsphere is provided, which is spherical; the interior of the shell is a cavity structure, the size of the cavity is 150 +/-10 nm, the exterior of the shell is a mesoporous silicon shell layer, and the thickness of the shell layer is 55 +/-5 nm.
Furthermore, the shell layer of the nano microsphere is provided with a mesoporous structure of 3 +/-1 nm.
Furthermore, the particle size of the nano-microsphere is 263 +/-19 nm.
Further, the specific surface area of the nano-microsphere is 885m2G, total pore volume 1.06cm3/g。
Furthermore, the structure of the nano-microsphere is stable, and the hollow structure of the nano-microsphere is stable after being calcined for 6 hours at 600 ℃.
The invention also provides a preparation method of the hollow mesoporous silicon nano microsphere.
The preparation method of the hollow mesoporous silicon nano microsphere comprises the following steps:
(1) weighing 0.8g of hexadecyl trimethyl ammonium bromide, adding a solvent, and stirring at room temperature for 1 hour to form a uniform solution;
(2) adding 10g of polystyrene microsphere solution and an alkaline catalyst into the solution obtained in the step (1), performing ultrasonic dispersion for 10min, continuously stirring for 30min, adding 3mL of silicon source, and reacting at room temperature for 48 h;
(3) performing solid-liquid separation on the solution in the step (2), and washing the solid phase with ethanol and water for 2-4 times;
(4) calcining the precipitate obtained in the step (3) for 6 hours at 600 ℃ to obtain the hollow mesoporous nano microsphere.
Further, the solvent in the step (1) is 29mL of water and 12mL of ethanol;
the alkaline catalyst is a 28% ammonia water solution, and the using amount is 1 mL.
Further, in the step (2), the silicon source is tetraethyl orthosilicate;
the polystyrene microsphere solution is an aqueous solution of polystyrene microspheres, and the concentration of the solution is 5% w/w;
and (4) performing solid-liquid separation in the step (3) by centrifugation at 10000rpm for 10 min.
The invention also provides the application of the hollow mesoporous silicon nano microsphere.
The hollow mesoporous silicon nano microsphere is used as a virus-like particle vaccine adjuvant.
The invention also provides a preparation method of the hollow mesoporous silicon nano microsphere as a virus-like particle vaccine adjuvant.
The preparation method of the hollow mesoporous silicon nano microsphere as the virus-like particle vaccine adjuvant comprises the following steps:
(1) PBS buffer solution is added into the nano-microspheres to prepare 5mg/mL solution;
(2) mixing the solution with 0.5mg/mL virus-like particle antigen according to a ratio of 1:1, and standing overnight at 4 ℃ to obtain the vaccine containing the nano microsphere adjuvant.
The hollow mesoporous silicon nano microsphere provided by the invention is prepared in ethanol and aqueous solution by taking tetraethyl orthosilicate as a silicon source, taking polystyrene microspheres and hexadecyl trimethyl ammonium bromide as templates and ammonia water as an alkaline catalyst.
The hollow mesoporous silicon nano microsphere provided by the invention has the characteristics of low toxicity, easy degradation, easy surface functionalization modification and the like. Compared with other nano materials, the huge specific surface area and specific pore volume of the nano material can adsorb and contain more proteins.
The hollow mesoporous silicon nano microspheres provided by the invention are used as adjuvants, are combined with foot-and-mouth disease virus-like particles according to a proportion to immunize guinea pigs, can induce organisms to generate effective immune response, have higher levels of specific antibodies and neutralizing antibodies, and have higher protection rate on the guinea pigs after being attacked by toxin.
The hollow mesoporous silicon nano microsphere has the advantages of good safety, high stability, simple preparation and the like, and can be used for preparing foot-and-mouth disease virus-like particle vaccines.
Compared with the prior art, the hollow mesoporous silicon nano microsphere and the preparation method and the application thereof provided by the invention have the advantages that:
(1) the hollow mesoporous silicon nano microsphere has the advantages of simple preparation method, high yield and uniform particle size, and is beneficial to the loading and slow release of antigen;
(2) the hollow mesoporous silicon nano microsphere has good biological safety, including good blood compatibility and tissue compatibility, is nontoxic to cells in a conventional dosage range, and has no obvious side reaction and side effect on experimental animals.
(3) The hollow mesoporous silicon nano microsphere is used as a foot-and-mouth disease virus-like particle vaccine adjuvant, can protect antigens, stimulates an organism to generate immune response, generates higher antibody level, and has higher protection effect on a guinea pig after being attacked by virus.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) image of the hollow mesoporous silicon nanospheres provided by the present invention.
FIG. 2 is a Scanning Electron Microscope (SEM) image of the hollow mesoporous silicon nanospheres provided by the invention.
FIG. 3 is a specific surface area diagram of the hollow mesoporous silicon nanospheres provided by the invention.
Fig. 4 shows the results of the test of neutralizing antibodies and specific antibodies of the hollow mesoporous silica nanospheres and the commercial vaccine adjuvant ISA 206 provided by the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not for limiting the scope of the present invention.
A hollow mesoporous silicon nanometer microsphere is provided, which is spherical; the interior of the shell is a cavity structure, the size of the cavity is 150 +/-10 nm, the exterior of the shell is a mesoporous silicon shell layer, and the thickness of the shell layer is 55 +/-5 nm.
Furthermore, the shell layer of the nano microsphere is provided with a mesoporous structure of 3 +/-1 nm.
Furthermore, the particle size of the nano-microsphere is 263 +/-19 nm.
Further, the specific surface area of the nano-microsphere is 885m2G, total pore volume 1.06cm3/g。
Furthermore, the structure of the nano-microsphere is stable, and the hollow structure of the nano-microsphere is stable after being calcined for 6 hours at 600 ℃.
The invention also provides a preparation method of the hollow mesoporous silicon nano microsphere.
The preparation method of the hollow mesoporous silicon nano microsphere comprises the following steps:
(1) weighing 0.8g of hexadecyl trimethyl ammonium bromide, adding a solvent, and stirring at room temperature for 1 hour to form a uniform solution;
(2) adding 10g of polystyrene microsphere solution and an alkaline catalyst into the solution obtained in the step (1), performing ultrasonic dispersion for 10min, continuously stirring for 30min, adding 3mL of silicon source, and reacting at room temperature for 48 h;
(3) performing solid-liquid separation on the solution in the step (2), and washing the solid phase with ethanol and water for 2-4 times;
(4) calcining the precipitate obtained in the step (3) for 6 hours at 600 ℃ to obtain the hollow mesoporous nano microsphere.
Further, the solvent in the step (1) is 29mL of water and 12mL of ethanol;
the alkaline catalyst is a 28% ammonia water solution, and the using amount is 1 mL.
Further, in the step (2), the silicon source is tetraethyl orthosilicate;
the polystyrene microsphere solution is an aqueous solution of polystyrene microspheres, and the concentration of the solution is 5% w/w;
and (4) performing solid-liquid separation in the step (3) by centrifugation at 10000rpm for 10 min.
The invention also provides the application of the hollow mesoporous silicon nano microsphere.
The hollow mesoporous silicon nano microsphere is used as a virus-like particle vaccine adjuvant.
The invention also provides a preparation method of the hollow mesoporous silicon nano microsphere as a virus-like particle vaccine adjuvant.
The preparation method of the hollow mesoporous silicon nano microsphere as the virus-like particle vaccine adjuvant comprises the following steps:
(1) PBS buffer solution is added into the nano-microspheres to prepare 5mg/mL solution;
(2) mixing the solution with 0.5mg/mL virus-like particle antigen according to a ratio of 1:1, and standing overnight at 4 ℃ to obtain the vaccine containing the nano microsphere adjuvant.
Example 1
Preparation of hollow mesoporous silicon nano microsphere
As shown in fig. 1 to 3.
(1) Weighing 0.8g of hexadecyl trimethyl ammonium bromide, adding 29mL of water and 12mL of ethanol, putting into a 100mL round-bottom flask, and stirring at room temperature for 1h to form a uniform solution;
(2) adding 10g of polystyrene microsphere solution (160 nm; 5%, w/w) and 1mL of ammonia water (28%), ultrasonically dispersing for 10min, continuously stirring for 30min, adding 3mL of tetraethyl orthosilicate, and reacting for 48h at room temperature;
(3) cooling to room temperature after the reaction is finished, centrifuging at 10000rpm for 10min, collecting precipitate, and washing the precipitate with ethanol and water for 3 times;
(4) calcining the obtained precipitate at 600 ℃ for 6h to obtain the hollow mesoporous silicon nano microsphere with the particle size of 263 nm.
The structure is shown in figure 1 (transmission electron microscope image of hollow mesoporous silicon nano microsphere) and figure 2 (scanning electron microscope image of hollow mesoporous silicon nano microsphere). The specific surface area is shown in fig. 3 (a specific surface area diagram of the hollow mesoporous silicon nanospheres).
Example 2
Preparation of foot-and-mouth disease virus-like particle vaccine
(1) Weighing 10mg of hollow mesoporous silicon nano microspheres, and adding 2mL of PBS buffer solution to prepare 5mg/mL solution;
(2) mixing the solution with 0.5mg/mL foot-and-mouth disease virus-like particle antigen according to a ratio of 1:1, and mixing overnight at 4 ℃ to obtain the vaccine containing the nano microsphere adjuvant.
Example 3
Guinea pig immunoassay
Guinea pigs were selected as test subjects, and 24 guinea pigs were divided into four groups of 6 animals each.
The first group served as a blank control, the second group was injected with 0.2mL of a vaccine containing 500. mu.g of Solid Silicon Nanospheres (SSNs) and 50. mu.g of antigen per guinea pig as a control, the third group was injected with 0.2mL of a vaccine containing 500. mu.g of Hollow Mesoporous Silicon Nanospheres (HMSNs) and 50. mu.g of antigen per guinea pig, and the fourth group was injected with 0.2mL of a vaccine containing the commercial adjuvant ISA 206 and 50. mu.g of antigen per guinea pig.
Blood is collected 56 days after immunization, serum is separated, and a neutralizing antibody and a specific antibody are respectively tested and subjected to a challenge protection experiment.
The test results are shown in table 1 and fig. 4.
TABLE 1 result of protection rate against toxic attack of hollow mesoporous silicon nanospheres
Animal experiments show that the specific antibody, the neutralizing antibody and the toxicity attack protection rate of the vaccine preparation containing the hollow mesoporous silicon nano microspheres are far higher than those of a blank control group and a control group, which indicates that the hollow mesoporous silicon nano microspheres can enhance the immune response level and improve the toxicity attack protection rate. Compared with the commercial adjuvant ISA 206, the hollow mesoporous silicon nano microsphere has an immune effect equivalent to that of the commercial adjuvant.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various changes may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are included in the protective scope of the present invention.
It should be noted that, in the foregoing embodiments, various specific technical features and steps described in the above embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features and steps are not described separately.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A hollow mesoporous silicon nanometer microsphere is characterized in that: the nano-microspheres are spherical;
the interior of the shell is a cavity structure, the size of the cavity is 150 +/-10 nm, the exterior of the shell is a mesoporous silicon shell layer, and the thickness of the shell layer is 55 +/-5 nm.
2. The hollow mesoporous silicon nanosphere of claim 1, wherein: the shell layer of the nano microsphere is provided with a mesoporous structure of 3 +/-1 nm.
3. The hollow mesoporous silicon nanosphere of claim 1, wherein: the particle size of the nano-microsphere is 263 +/-19 nm.
4. The hollow mesoporous silicon nanosphere of claim 1, wherein: the specific surface area of the nano-microsphere is 885m2G, total pore volume 1.06cm3/g。
5. The hollow mesoporous silicon nanosphere of claim 1, wherein: the structure of the nano-microsphere is stable, and the hollow structure of the nano-microsphere is stable after being calcined for 6 hours at 600 ℃.
6. The preparation method of the hollow mesoporous silicon nanospheres according to any one of claims 1 to 5, wherein the preparation method comprises the following steps:
(1) weighing 0.8g of hexadecyl trimethyl ammonium bromide, adding a solvent, and stirring at room temperature for 1 hour to form a uniform solution;
(2) adding 10g of polystyrene microsphere solution and an alkaline catalyst into the solution obtained in the step (1), performing ultrasonic dispersion for 10min, continuously stirring for 30min, adding 3mL of silicon source, and reacting at room temperature for 48 h;
(3) performing solid-liquid separation on the solution in the step (2), and washing the solid phase with ethanol and water for 2-4 times;
(4) calcining the precipitate obtained in the step (3) for 6 hours at 600 ℃ to obtain the hollow mesoporous nano microsphere.
7. The preparation method of the hollow mesoporous silicon nanospheres according to claim 6, wherein the preparation method comprises the following steps: the solvent in the step (1) is 29mL of water and 12mL of ethanol;
the alkaline catalyst is a 28% ammonia water solution, and the using amount is 1 mL.
8. The preparation method of the hollow mesoporous silicon nanospheres according to claim 6, wherein the preparation method comprises the following steps: in the step (2), the silicon source is tetraethyl orthosilicate;
the polystyrene microsphere solution is an aqueous solution of polystyrene microspheres, and the concentration of the solution is 5% w/w;
and (4) performing solid-liquid separation in the step (3) by centrifugation at 10000rpm for 10 min.
9. The use of the hollow mesoporous silicon nanospheres according to any of claims 1 to 5, wherein the hollow mesoporous silicon nanospheres comprise: the nano-microsphere is used as a virus-like particle vaccine adjuvant.
10. The application of the hollow mesoporous silicon nanospheres according to claim 9, wherein the preparation method of the nanospheres as the virus-like particle vaccine adjuvant comprises:
(1) PBS buffer solution is added into the nano-microspheres to prepare 5mg/mL solution;
(2) mixing the solution with 0.5mg/mL virus-like particle antigen according to a ratio of 1:1, and standing overnight at 4 ℃ to obtain the vaccine containing the nano microsphere adjuvant.
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| CN105923636A (en) * | 2016-04-13 | 2016-09-07 | 哈尔滨工业大学 | Preparation method of monodisperse hollow mesoporous silica nanoparticle |
| WO2019028387A1 (en) * | 2017-08-03 | 2019-02-07 | Rita Elena Serda | Liposomal coated nanoparticles for immunotherapy applications |
| CN111601771A (en) * | 2017-11-14 | 2020-08-28 | 英国N4制药有限公司 | Process for producing particulate material |
| CN108275687A (en) * | 2018-03-29 | 2018-07-13 | 东北林业大学 | A kind of preparation method of mesoporous hollow silica microsphere |
| CN109796019A (en) * | 2019-02-21 | 2019-05-24 | 华中科技大学 | A kind of hollow silicon dioxide nanosphere and its preparation method and application |
| CN111658767A (en) * | 2019-06-13 | 2020-09-15 | 四川大学 | Hydrophilic antigen and/or hydrophobic antigen vaccine delivery system and preparation method thereof |
| JP2021175741A (en) * | 2020-04-28 | 2021-11-04 | 東洋製罐グループホールディングス株式会社 | Inorganic antiviral agent |
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