CN114128723B - Antiviral nano material and application thereof - Google Patents
Antiviral nano material and application thereof Download PDFInfo
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- CN114128723B CN114128723B CN202111322681.XA CN202111322681A CN114128723B CN 114128723 B CN114128723 B CN 114128723B CN 202111322681 A CN202111322681 A CN 202111322681A CN 114128723 B CN114128723 B CN 114128723B
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Abstract
The invention discloses a novel antiviral nanomaterial and application thereof. Specifically, the antiviral nanomaterial is nanosilicon dioxide (nSiO) 2 ). By the inventionDesigned multiple experiments, (1) nSiO 2 Co-incubating the reinfected cells with the viral particles; (2) nSiO 2 Treating cells and then infecting viruses; (3) Virus first infects cells and then nSiO 2 The cells were treated and the results confirmed that nSiO 2 After the cells are treated, the infection of the cells by viruses can be inhibited, and the virus particles and nSiO 2 Infection of cells after co-incubation results in poor viral infection.
Description
Technical Field
The invention belongs to the medicine technology, and in particular relates to a novel antiviral nano material and a preparation method and application thereof.
Background
In nature, viruses are of a wide variety, including DNA viruses, RNA viruses, prions, and the like, and almost all organisms have infectious virus types. Due to the characteristics of various viruses, high mutation speed and the like, only a few types of viruses are analyzed. Due to factors such as complex virus infection process, few specific and targeted medicines exist at present. The existing antiviral drugs are usually only aimed at single or certain viruses, and are required to be continuously taken for resisting viruses. Thus, the development of broad-spectrum antiviral nanoparticles is urgent.
Disclosure of Invention
The invention discloses a novel antiviral nano material nano silicon dioxide (nSiO) 2 ) Multiple experiments were designed, (1) nSiO 2 Co-incubating the reinfected cells with the viral particles; (2) nSiO 2 Treating cells and then infecting viruses; (3) Virus first infects cells and then nSiO 2 Cells are treated. Research results prove that nSiO 2 After the cells are treated, the virus pair can be inhibitedInfection of cells, while virions and nSiO 2 Infection of cells after co-incubation results in poor viral infection.
The invention adopts the following technical scheme:
the application of nano silicon dioxide as an antiviral nano material.
The invention discloses a method for improving antiviral ability of cells by utilizing nano particles, which comprises the steps of incubating the nano particles with the cells to improve the antiviral ability of the cells; or incubating the nano particles with the cells infected by the virus to improve the antiviral capability of the cells; the nano particles are nano silicon dioxide.
The invention discloses a method for reducing the capability of virus to infect cells by utilizing nano particles, which comprises the steps of mixing the nano particles with the virus to reduce the capability of the virus to infect the cells; the nano particles are nano silicon dioxide. In practical application, the nano silicon dioxide can be sprayed in an environment with viruses, so that the invasion of the viruses to cells is reduced.
Preferably, the virus is a DNA virus, RNA virus or prion; as a specific example, the virus is a reovirus, in particular an aquatic animal reovirus, such as grass carp reovirus (Grass Carp Reovirus, GCRV).
In the present invention, the cells are conventional cells.
Preferably, incubating the nanoparticle solution with the cells to increase antiviral ability of the cells; or incubating the nanoparticle solution with the virus-infected cells to improve the antiviral ability of the cells.
Preferably, the nanoparticle solution is mixed with the virus to reduce the ability of the virus to infect cells.
In the present invention, when the nanosilica is used as an antiviral nanomaterial, it is preferable that the nanosilica is present as a nanosilica solution, and the concentration of the nanosilica solution is from 0.5 to 10. Mu.g/mL, preferably from 0.75 to 8. Mu.g/mL, most preferably from 3 to 7. Mu.g/mL, such as from 5 to 6. Mu.g/mL. The solvent (alternatively referred to as a dispersing agent) in the nanosilica solution is selected according to the application, such as incubation with cells using a culture medium as the silica dispersing medium.
In the present invention, the particle size of the nanosilica is 1 to 100nm, preferably 5 to 50nm, such as 10 to 30nm.
The nano technology is a interdisciplinary research field, and the nano material has application in the aspects of antibacterial drug substitution, packaging materials and plant disease sterilization due to excellent physical and chemical properties, high specific surface area and nano structure, and particularly has the antibacterial activity due to the interaction of the larger specific surface area and pathogenic bacteria. So far, the action mechanism of nano materials, especially nano silicon dioxide, on viruses is not clear, and different nano materials have differences of application effects on different viruses. Multiple experiments were designed, (1) nSiO 2 Co-incubating the reinfected cells with the viral particles; (2) nSiO 2 Treating cells and then infecting viruses; (3) Virus first infects cells and then nSiO 2 Cells are treated. Research results prove that nSiO 2 After the cells are treated, the infection of the cells by viruses can be inhibited, and the virus particles and nSiO 2 Infection of cells after co-incubation results in poor viral infection.
Drawings
FIG. 1 is a scanning electron microscope image of nano silicon dioxide, nano SiO 2 Dispersing in absolute ethanol (concentration is 50 ug/mL), and the testing instrument is Hitachi S-4700 cold field emission scanning electron microscope.
FIG. 2 is a scanning electron microscope image of nano silicon dioxide, nano SiO 2 The powder and the testing instrument are Hitachi SU-8230 field emission scanning electron microscope.
FIG. 3 is an infrared spectrum of nano-silica.
FIG. 4 shows the toxicity of nanosilica at different concentrations to CIK cells.
FIG. 5 nSiO concentration of different concentrations 2 Effects of the treated cells on the expression level of the viral nonstructural protein NS 4.
FIG. 6 is a graph of nSiO concentration 2 Effects of the treated cells on the expression level of the viral structural protein VP 7.
FIG. 7 is nSiO 2 Different treatments were used for the cell infection.
FIG. 8 is nSiO 2 Effect of different treatments on expression level of GCRV structural protein VP7 protein, a is gel electrophoresis pattern and B is relative quantification pattern.
Detailed Description
The nanostructure consists of nanoscale ultrafine particles with a defined morphology, which have higher activity than large particle materials due to their larger surface area and size effects. The invention uses the existing nano silicon dioxide (nSiO) 2 ) For example, multiple sets of experiments were designed for grass carp kidney Cells (CIK) and grass carp reoviruses (Grass Carp Reovirus, GCRV), demonstrating their antiviral ability; the related nanometer materials, culture medium, viruses, cells, electrophoresis materials and the like are all conventional products, and the specific operation method and the testing method are all conventional technologies.
Fig. 1 and 2 are nano silicon dioxide scanning electron microscope diagrams, and fig. 3 is a nano silicon dioxide infrared spectrogram; it can be seen that SiO 2 Spherical particles with uniform size and particle diameter of about 10-20 nm, and the nano SiO 2 Zeta potential (zeta potential) in deionized water (50. 50 ug/mL) was-26.12 mV, and zeta potential in working fluid at 6. 6 ug/mL was-8.93 mV.
Example MTT method for detecting toxicity of nanomaterial
(1) Preparing MTT solution: weighing MTT0.5g, dissolving in 100mL phosphate buffer solution 1 XPBS (pH 7.4), filtering with 0.22 μm filter membrane, packaging, and preserving at-20deg.C to avoid repeated freezing and thawing;
(2) CIK cells were collected and the cell suspension concentration was adjusted to 1X 10 5 1 96-well plate was taken per mL, 200. Mu.L of cell suspension was added to each well of the 96-well plate, the edges of the 96-well plate were filled with sterile 1 XPBS to eliminate edge effects, and incubated at 26℃for 12h;
(3) Dissolving nano SiO with fresh 1640 culture medium containing 10% FBS 2 The final concentrations were set to 0. Mu.g/mL, 0.75. Mu.g/mL, 1.5. Mu.g/mL, 3. Mu.g/mL, and 6. Mu.g/mL, respectively;
(4) Every five compound holes in the 96-well plate are set as a group, and the nano SiO diluted in the previous step is respectively added into each group of cells 2 Culturing at 26 ℃ for 6 hours;
(5) 20 mu LMTT solution (5 mg/mL, i.e., 0.5% MTT) was added to each well and incubation was continued for 4h;
(6) Terminating the culture, and carefully sucking out the culture solution in the hole;
(7) Adding 150 mu L of dimethyl sulfoxide into each hole, placing on a shaking table, shaking for 15min, and measuring the absorbance value of each hole at the OD490nm position of an ELISA (enzyme-linked immunosorbent assay) after the crystal is fully dissolved;
(8) Zero-setting wells (medium, MTT, dimethyl sulfoxide) were set.
Referring to fig. 4, the nano-silica working solutions of different concentrations according to the present invention have little toxicity to CIK cells.
Example second qPCR method to test the inhibition of the virus by nanomaterials
1mL of nano SiO with different concentrations 2 (0. Mu.g/mL, 0.75. Mu.g/mL, 1.5. Mu.g/mL, 3. Mu.g/mL, 6. Mu.g/mL, dispersion medium was conventional CIK cell culture medium) working solution was mixed with 2ul of GCRV (MOI=4) at 26℃for 1 hour, followed by 1X 10 additions 6 The CIK cells were allowed to stand at 26℃for 1h, followed by pipetting out the nano SiO 2 Mixing with virus mixture, and culturing for 48 hr; then total RNA of the cells was extracted, and after digesting the genomic DNA with DNaseI, it was reverse transcribed into cDNA using random primers, and then the expression level of NS4 gene was detected by real-time PCR using the primers shown in Table 1 (EF 1a-F is an internal reference), and the real-time PCR system and procedure were set as follows:
(1) Reaction system (20 mu L)
(2) The PCR cycle is as follows: after 10 min at 95.0deg.C, amplifying for 40 cycles at 95.0deg.C for 15 s and 55 deg.C for 30s, and fluorescent detection reading plate is set at 45 s stage at 55.0deg.C, and the dissolution curve is detected after the PCR reaction is completed, see FIG. 5.
Example three electrophoresis detection of nanoparticle inhibition of viral proteins
1mL of nano SiO with different concentrations 2 (0. Mu.g/mL, 0.75. Mu.g/mL, 1.5. Mu.g/mL, 3. Mu.g/mL, 6. Mu.g/mL, dispersion medium was conventional CIK cell culture medium) was mixed with 2ul of GCRV (MOI=4) at 26℃for 1h, followed by 1X 10 additions 6 In the CIK cells, standing at 26 ℃ for 1h, and then absorbing and discarding nano SiO 2 With virus mixture, culture was continued for 48 hours, and then cellular proteins were extracted for conventional gel electrophoresis, see FIG. 6.
Example four SiO 2 Is to influence the GCRV virus by different treatment modes
SiO 2 +gcrv:1mL (concentration of 6. Mu.g/mL) of nano SiO 2 Incubation with 2ul GCRV (moi=4) at 26 ℃ for 1h followed by 1×10 additions 6 In the CIK cells, standing at 26 ℃ for 1h, and then absorbing and discarding nano SiO 2 Mixing with virus mixture, and culturing; and then extracting the cell proteins for conventional gel electrophoresis, and observing the cell infection.
SiO 2 -:1mL (concentration of 6. Mu.g/mL) of nano SiO 2 Adding 1×10 6 After incubating the cells at 26 ℃ for 1 hour in the CIK cells, incubating the cells at 2ul GCRV (MOI=4) at 26 ℃ for 1 hour, sucking out the virus, and continuing to culture; and then extracting the cell proteins for conventional gel electrophoresis, and observing the cell infection.
SiO 2 +:2ul GCRV (MOI=4) after incubation of cells at 26℃for 1h, the virus was aspirated, 1mL (concentration 6. Mu.g/mL) of nano SiO 2 Incubating the cells at 26 ℃ for 1h, sucking out, and continuously culturing; and then extracting the cell proteins for conventional gel electrophoresis, and observing the cell infection.
FIG. 7 shows nSiO at the same incubation time 2 Different treatments are used for the infection of cells; FIG. 8 shows nSiO at the same incubation time 2 Effects of different treatments on expression levels of the GCRV structural protein VP7 protein. Substitution of nano SiO with nano zinc oxide of similar size 2 According to the formula SiO 2 The +GCRV group experiment step finds that the inhibition effect of nano zinc oxide on the expression level of GCRV structural protein VP7 protein and SiO 2 "group approximation, significantly lower than" SiO 2 +gcrv "group.
The invention adopts routine testMethod characterization of nanosilicon dioxide compatibility with Normal cells and Virus inhibition Capacity, first, nSiO was formulated at a concentration of 100. Mu.g/mL 2 The culture medium solution, diluted to various final concentrations (0.75, 1.5, 3 and 6. Mu.g/mL) treated cells 48h, was found to be very low in CIK cytotoxicity; then different final concentrations of nSiO 2 Mixing the culture medium solution with viruses, then incubating the viruses with the cells, sucking out the viruses, continuously culturing 48h, centrifuging to collect cell precipitates, collecting the cell precipitates, extracting total RNA in the cells by using an RNA extraction kit for one part, extracting total protein by using a protein extraction kit for the other part, quantifying the RNA and the protein by using a Nanodrop 2000 micro-ultraviolet spectrophotometer, carrying out SDS-PAGE separation on 20 mu g of protein, detecting the expression level of virus structural protein VP7 by using western blotting, carrying out reverse transcription on 2 mu g of RNA, and carrying out real-time PCR on the expression level of virus non-structural protein gene NS4, so that the nano silicon dioxide has an inhibition effect on VP7 and NS 4; finally comparing SiO 2 The method is optimal in that nano silicon dioxide and viruses are mixed first and then incubated with cells on the basis that the three methods have good effects on the influence of different treatment methods of GCRV viruses. Thus, according to the teachings of the present invention, nanosilica can be sprayed in an environment with viruses, thereby reducing the invasion of cells by the viruses.
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Claims (3)
1. The application of nano silicon dioxide in preparing an antiviral agent is characterized in that the virus is grass carp reovirus; the nano silicon dioxide exists in the form of nano silicon dioxide solution, and the concentration of the nano silicon dioxide solution is 0.5-10 mug/mL; the particle size of the nano silicon dioxide is 1-100 nm.
2. Use of nanoparticles for the preparation of a reagent for increasing antiviral ability of a cell, characterized in that the nanoparticles are incubated with the cell to increase antiviral ability of the cell; or incubating the nano particles with the cells infected by the virus to improve the antiviral capability of the cells; the nano particles are nano silicon dioxide; the virus is grass carp reovirus; the nano silicon dioxide exists in the form of nano silicon dioxide solution, and the concentration of the nano silicon dioxide solution is 0.5-10 mug/mL; the particle size of the nano silicon dioxide is 5-60 nm.
3. Use of a nanoparticle for the preparation of an agent that reduces the ability of a virus to infect a cell, wherein the nanoparticle is admixed with the virus to reduce the ability of the virus to infect the cell; the nano particles are nano silicon dioxide; the virus is grass carp reovirus; the nano silicon dioxide exists in the form of nano silicon dioxide solution, and the concentration of the nano silicon dioxide solution is 0.5-10 mug/mL; the particle size of the nano silicon dioxide is 5-60 nm.
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