WO2023082118A1 - 一种新型抗病毒纳米材料及其应用 - Google Patents

一种新型抗病毒纳米材料及其应用 Download PDF

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WO2023082118A1
WO2023082118A1 PCT/CN2021/129925 CN2021129925W WO2023082118A1 WO 2023082118 A1 WO2023082118 A1 WO 2023082118A1 CN 2021129925 W CN2021129925 W CN 2021129925W WO 2023082118 A1 WO2023082118 A1 WO 2023082118A1
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virus
cells
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胡小龙
张星
梁子
沈泽恩
朱天辰
贡成良
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苏州大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof

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  • the invention belongs to pharmaceutical technology, and in particular relates to a novel antiviral nanometer material and its preparation method and application.
  • viruses In nature, there are many kinds of viruses, including DNA viruses, RNA viruses, prions, etc., and almost all organisms have infected virus types. Due to the wide variety of viruses and the rapid mutation speed, only a very small number of viruses have been analyzed. Due to factors such as the complexity of the virus infection process, currently there are few specific and targeted drugs. Existing antiviral drugs often can only target a single virus or a certain type of virus, and need to be taken continuously to resist the virus. Therefore, it is imminent to develop broad-spectrum antiviral nanoparticles.
  • the invention discloses a new type of anti-viral nano-material nano-silica (nSiO 2 ), and several sets of experiments are designed, (1) nSiO 2 and virus particles are co-incubated to infect cells; (2) nSiO 2 first treats the cells, Re-infect the virus; (3) The virus first infects the cells, and then treats the cells with nSiO 2 .
  • the results of the study confirmed that nSiO 2 can inhibit virus infection of cells after treatment of cells, but the effect of virus infection on cells after co-incubation with virus particles and nSiO 2 is very weak.
  • 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 the anti-virus ability of cells by using nanoparticles.
  • the nano-particles are incubated with cells to improve the anti-virus ability of cells; For nano silicon dioxide.
  • the invention discloses a method for reducing the ability of virus to infect cells by using nanoparticles.
  • the nanoparticles are mixed with viruses to reduce the ability of viruses to infect cells; the nanoparticles are nano silicon dioxide.
  • nano-silica can be sprayed in an environment with viruses to reduce the virus's invasion of cells.
  • the virus is a DNA virus, RNA virus or prion; as a specific example, the virus is a reovirus, specifically an aquatic animal reovirus, such as Grass Carp Reovirus (GCRV).
  • GCRV Grass Carp Reovirus
  • cells are conventional cells.
  • the nanoparticle solution is incubated with the cells to improve the anti-virus ability of the cells; or the nanoparticle solution is incubated with the virus-infected cells to improve the anti-virus ability of the cells.
  • mixing the nanoparticle solution with the virus reduces the ability of the virus to infect cells.
  • nano-silica when used as an antiviral nano-material, it exists in the form of nano-silica solution.
  • concentration of nano-silica solution is 0.5-10 ⁇ g/mL, preferably 0.75-8 ⁇ g/mL , most preferably 3-7 ⁇ g/mL, such as 5-6 ⁇ g/mL.
  • the solvent (or dispersant) in the nano-silica solution is selected according to the application.
  • culture medium is used as the silica dispersion medium for co-incubation with cells.
  • the particle size of the nano silicon dioxide is 1-100 nm, preferably 5-50 nm, such as 10-30 nm.
  • Nanotechnology is an interdisciplinary research field. Due to its excellent physical and chemical properties, high specific surface area, and nanostructure, nanomaterials have applications in antibacterial drug substitution, packaging materials, and plant disease sterilization, especially when the large specific surface area interacts with pathogenic bacteria. role, has antibacterial activity. So far, the mechanism of action of nanomaterials, especially nanosilica, on viruses has not been clear, and different nanomaterials have different application effects on different viruses.
  • the present invention discloses the application of nanosilica as an antiviral nanomaterial for the first time.
  • Figure 1 is a scanning electron microscope image of nano-silica. Nano-SiO 2 is dispersed in absolute ethanol (concentration: 50 ug/mL). The testing instrument is a Hitachi S-4700 cold field emission scanning electron microscope.
  • Figure 2 is a scanning electron microscope image of nano-silica, nano-SiO 2 powder, and the testing instrument is a Hitachi SU-8230 field emission scanning electron microscope.
  • Fig. 3 is the nano-silica infrared spectrogram.
  • Figure 4 shows the toxicity of different concentrations of nano-silica to CIK cells.
  • Fig. 6 is the effect of different concentrations of nSiO 2 treating cells on the expression level of viral structural protein VP7.
  • Figure 7 shows the infection of cells by different treatments of nSiO 2 .
  • Figure 8 is the effect of different treatments of nSiO2 on the expression level of GCRV structural protein VP7 protein, A is the gel electrophoresis picture, and B is the relative quantitative picture.
  • Nanostructures are composed of nanoscale ultrafine particles with well-defined morphology, and due to larger surface area and size effect, nanostructures are more active than large particle materials.
  • nSiO 2 nano silicon dioxide
  • the present invention designs several groups of experiments on grass carp kidney cells (CIK) and grass carp reovirus (GCRV) to illustrate its antiviral ability; Materials, culture medium, viruses, cells, electrophoretic materials, etc. are all existing conventional products, and the specific operation methods and testing methods are conventional technologies.
  • Figure 1 and Figure 2 are scanning electron microscope images of nano-silica, and Figure 3 is the infrared spectrum of nano-silica; it can be seen that SiO 2 is basically spherical particles with uniform size and a particle diameter of about 10-20 nm.
  • the zeta potential ( ⁇ potential) of SiO 2 in deionized water (50 ug/mL concentration) is -26.12 mV, and the zeta potential is -8.93 mV in the working solution with a concentration of 6 ug/mL.
  • Example 1 MTT method to detect the toxicity of nanomaterials (1) Preparation of MTT solution: Weigh 0.5g of MTT, dissolve it in 100mL phosphate buffer solution 1 ⁇ PBS (pH 7.4), filter it with a 0.22 ⁇ m filter membrane, and dispense it at -20 Store at °C, avoid repeated freezing and thawing.
  • the nano-silica working solution with different concentrations of the present invention has little toxicity to CIK cells.
  • SiO 2 +: 2ul GCRV (MOI 4) incubate the cells at 26°C for 1 hour and then suck out the virus, 1mL (concentration of 6 ⁇ g/mL) nano-SiO 2 incubate the cells at 26°C for 1 hour, then suck out the cells, continue to culture; then extract the cell protein for routine coagulation Gel electrophoresis to observe the infection of cells.
  • Figure 7 shows the infection of cells by different treatments of nSiO2 under the same culture time
  • Figure 8 shows the effect of different treatments of nSiO2 on the expression level of GCRV structural protein VP7 protein under the same culture time.
  • the present invention uses conventional test methods to characterize the compatibility of nano silicon dioxide to normal cells and the ability to inhibit viruses.
  • a nSiO2 medium solution with a concentration of 100 ⁇ g/mL is prepared and diluted to different final concentrations (0.75, 1.5, 3 and 6 ⁇ g/mL) to treat cells for 48 h, and found that the toxicity to CIK cells was very low; then mixed different final concentrations of nSiO 2 medium solution with the virus and then incubated with the cells, sucked out the virus, continued to culture for 48 h, and collected the cells by centrifugation Precipitation, the collected cell pellets, part of the total RNA in the cells was extracted with an RNA extraction kit, and the other part was extracted with a protein extraction kit for total protein, and RNA and protein were quantified with a NanoDrop 2000 micro-volume ultraviolet spectrophotometer, and 20 ⁇ g of protein was taken for SDS-PAGE separation, and western blotting was used to detect the expression level of the viral structural
  • Silicon oxide has an inhibitory effect on VP7 and NS4; finally, the effects of different treatment methods of SiO 2 on GCRV virus were compared. On the basis that all three methods have good effects, nano-silica and virus were first mixed and then incubated with cells the best way. Therefore, according to the revelation of the present invention, nano-silica can be sprayed in an environment with viruses, thereby reducing the virus's invasion of cells.

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Abstract

一种新型抗病毒纳米材料及其应用，抗病毒纳米材料为纳米二氧化硅(nSiO 2)。设计多组实验，(1)nSiO 2和病毒粒子共孵育再感染细胞；(2)nSiO 2先处理细胞，再感染病毒；(3)病毒先感染细胞，再nSiO 2处理细胞，结果证实nSiO 2处理细胞后，能够抑制病毒对细胞的感染，而病毒粒子和nSiO 2共孵育后再感染细胞则病毒感染效果很弱。

Description

一种新型抗病毒纳米材料及其应用 技术领域
本发明属于药物技术，具体涉及一种新型抗病毒纳米材料及其制备方法与应用。
 背景技术
自然界，病毒种类繁多，有DNA病毒、RNA病毒、朊病毒等，且几乎所有的生物都有感染的病毒类型。由于病毒种类繁多，变异速度快等特点，只要极少数种类的病毒得到了解析。由于病毒感染过程复杂等因素，目前具有特效的、针对性的药物还是较少。现有的抗病毒药物往往只能针对单一的或者某一类病毒，而且需要持续服用来抵抗病毒。因此研究出广谱型抗病毒纳米颗粒迫在眉睫。
 技术问题
本发明公开了一种新型的抗病毒纳米材料纳米二氧化硅（nSiO 2）,设计了多组实验，（1）nSiO 2和病毒粒子共孵育再感染细胞；(2)nSiO 2先处理细胞，再感染病毒；（3）病毒先感染细胞，再nSiO 2处理细胞。研究结果证实nSiO 2处理细胞后，能够抑制病毒对细胞的感染，而病毒粒子和nSiO 2共孵育后再感染细胞则病毒感染效果很弱。
技术解决方案
本发明采用如下技术方案：纳米二氧化硅作为抗病毒纳米材料的应用。
本发明公开了一种利用纳米颗粒提高细胞抗病毒能力的方法，将纳米颗粒与细胞孵育，提高细胞抗病毒能力；或者将纳米颗粒与病毒感染后的细胞孵育，提高细胞抗病毒能力；纳米颗粒为纳米二氧化硅。
本发明公开了一种利用纳米颗粒降低病毒感染细胞能力的方法，将纳米颗粒与病毒混合，降低病毒感染细胞的能力；纳米颗粒为纳米二氧化硅。实际应用时，可在带有病毒的环境中，喷洒纳米二氧化硅，从而降低病毒对细胞的侵袭。
优选的，病毒为DNA病毒、RNA病毒或者朊病毒；作为具体的示例，病毒为呼肠孤病毒，具体为水生动物呼肠孤病毒，比如草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)。
本发明中，细胞为常规细胞。
优选的，将纳米颗粒溶液与细胞孵育，提高细胞抗病毒能力；或者将纳米颗粒溶液与病毒感染后的细胞孵育，提高细胞抗病毒能力。
优选的，将纳米颗粒溶液与病毒混合，降低病毒感染细胞的能力。
本发明中，纳米二氧化硅作为抗病毒纳米材料的应用时，以纳米二氧化硅溶液形式存在，优选的，纳米二氧化硅溶液的浓度为0.5～10μg/mL，优选为0.75～8μg/mL，最优选为3～7μg/mL，比如5～6μg/mL。纳米二氧化硅溶液中的溶剂（或者称为分散剂）根据应用选择，比如与细胞共孵育则采用培养基作为二氧化硅分散介质。
本发明中，纳米二氧化硅的粒径为1～100nm，优选为5～50nm，比如10～30nm。
 有益效果
纳米技术为跨学科的研究领域，纳米材料由于卓越的物理化学性能、高比表面积以及纳米结构，在抗菌药物替代、包装材料、植物病害杀菌方面有应用，尤其是较大的比表面积与病原菌相互作用，具有抗菌活性。目前为止，纳米材料尤其是纳米二氧化硅对病毒的作用机制还未清楚，并且不同纳米材料对不同病毒具有应用效果的差异，本发明首次公开了纳米二氧化硅作为抗病毒纳米材料的应用。设计了多组实验，（1）nSiO 2和病毒粒子共孵育再感染细胞；(2)nSiO 2先处理细胞，再感染病毒；（3）病毒先感染细胞，再nSiO 2处理细胞。研究结果证实nSiO 2处理细胞后，能够抑制病毒对细胞的感染，而病毒粒子和nSiO 2共孵育后再感染细胞则病毒感染效果很弱。
附图说明
图1为纳米二氧化硅扫描电镜图，纳米SiO 2分散于无水乙醇中（浓度为50 ug/mL），测试仪器为日立S-4700冷场发射扫描电子显微镜。
图2为纳米二氧化硅扫描电镜图，纳米SiO 2粉末，测试仪器为日立SU-8230场发射扫描电子显微镜。
图3为纳米二氧化硅红外光谱图。
图4为不同浓度纳米二氧化硅对CIK细胞的毒性。
图5不同浓度的nSiO 2处理细胞对病毒非结构蛋白NS4表达水平的影响。
图6为不同浓度的nSiO 2处理细胞对病毒结构蛋白VP7表达水平的影响。
图7为nSiO 2不同处理对细胞感染情况。
图8为nSiO 2不同处理对GCRV结构蛋白VP7蛋白表达水平的影响，A为凝胶电泳图，B为相对定量图。
本发明的实施方式
纳米结构由具有明确形态的纳米尺度超细颗粒组成的，由于具有更大的表面积和尺寸效应，纳米结构比大颗粒材料具有更高的活性。本发明以现有纳米二氧化硅（nSiO 2）为例，针对草鱼肾细胞（CIK）与草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)设计多组实验，说明其抗病毒能力；涉及的纳米材料、培养基、病毒、细胞、电泳材料等都是现有常规产品，具体操作方法以及测试方法都为常规技术。
图1、图2为纳米二氧化硅扫描电镜图，图3为纳米二氧化硅红外光谱图；可以看到，SiO 2基本呈球状颗粒，大小均匀，粒径约为10～20 nm，该纳米SiO 2在去离子水中（浓度为50 ug/mL）的zeta电位（ζ potential）为-26.12 mV，在浓度为6 ug/mL的工作液中，zeta电位为-8.93 mV。
实施例一 MTT法检测纳米材料毒性：（1）MTT溶液配制：称取MTT0.5g，溶于100mL磷酸缓冲液1×PBS（pH 7.4）中，用0.22μm滤膜过滤后分装，-20℃保存，避免反复冻融。
（2）收集CIK细胞，调整细胞悬液浓度为1×10 5个/mL，取1块96孔板，向96孔板中每孔加入200μL细胞悬液，96孔板边缘用无菌的1×PBS填充以消除边缘效应，26℃培养12h。
（3）用新鲜的含10% FBS的1640培养基溶解纳米SiO 2，使其终浓度分别为0μg/mL、0.75μg/mL、1.5μg/mL、3μg/mL、6μg/mL。
（4）96孔板中每五个复孔设为一组，向每组细胞中分别加入上一步稀释好的纳米SiO 2，26℃培养6h。
（5）每孔加入20μLMTT溶液（5mg/mL，即0.5%MTT），继续培养4h。
（6）终止培养，小心吸去孔内培养液。
（7）每孔加入150μL二甲基亚砜，置于摇床上震荡15min，待结晶物充分溶解后，在酶联免疫检测仪OD490nm处测量各孔的吸光值。
（8）设置调零孔（培养基、MTT、二甲基亚砜）。
参见图4，本发明不同浓度纳米二氧化硅工作液对CIK细胞的毒性不大。
实施例二 qPCR方法测试纳米材料对病毒的抑制：将1mL不同浓度的纳米SiO 2（0μg/mL、0.75μg/mL、1.5μg/mL、3μg/mL、6μg/mL，分散介质为常规CIK细胞培养基）工作液与2ul GCRV（MOI=4）置于26℃混合1h，随后加入1×10 6个CIK细胞，26℃静止1h，随后吸弃纳米SiO 2与病毒混合液，继续培养48h；然后提取细胞总RNA，用DNaseI消化基因组DNA后，用随机引物反转录成cDNA，随后用表1所列引物通过real-time PCR检测NS4基因的表达水平（EF1a-F为内参），real-time PCR体系和程序设定如下：（1）反应体系（20 µL）
Figure 515052dest_path_image001
。
（2）PCR循环的过程为：95.0℃ 10 min 后，按95.0℃ 15 s，55℃ 30s扩增40个循环，荧光检测读板设置在55.0℃ 45 s阶段，PCR反应结束后检测溶解曲线，参见图5。
Figure 560369dest_path_image002
。
实施例三 电泳检测纳米颗粒对病毒蛋白的抑制：将1mL不同浓度的纳米SiO 2（0μg/mL、0.75μg/mL、1.5μg/mL、3μg/mL、6μg/mL，分散介质为常规CIK细胞培养基）与2ul GCRV（MOI=4）置于26℃混合1h，随后加入1×10 6个CIK细胞中，26℃静置1h，随后吸弃纳米SiO 2与病毒混合液，继续培养48h，然后提取细胞蛋白进行常规凝胶电泳，参见图6。
实施例四 SiO 2的不同处理方式对GCRV病毒的影响：SiO 2+GCRV：1mL（浓度为6μg/mL）的纳米SiO 2与2ul GCRV（MOI=4）26 ℃共孵育1h后加入1×10 6个CIK细胞中，26℃静置1h，随后吸弃纳米SiO 2与病毒混合液，继续培养；然后提取细胞蛋白进行常规凝胶电泳，观察细胞感染情况。
SiO 2-：1mL（浓度为6μg/mL）纳米SiO 2加入1×10 6个CIK细胞中，26℃孵育细胞1h后，2ul GCRV（MOI=4）26℃孵育细胞1h，将病毒吸出，继续培养；然后提取细胞蛋白进行常规凝胶电泳，观察细胞感染情况。
SiO 2+：2ul GCRV（MOI=4）26℃孵育细胞1h后将病毒吸出，1mL（浓度为6μg/mL）纳米SiO 2 26℃孵育细胞1h后吸出，继续培养；然后提取细胞蛋白进行常规凝胶电泳，观察细胞感染情况。
图7为同样培养时间下，nSiO 2不同处理对细胞感染情况；图8为同样培养时间下，nSiO 2不同处理对GCRV结构蛋白VP7蛋白表达水平的影响。以大小近似的纳米氧化锌替换纳米SiO 2，按“SiO 2+GCRV”组实验步骤，发现纳米氧化锌对GCRV结构蛋白VP7蛋白表达水平的抑制效果与“SiO 2-”组近似，明显低于“SiO 2+GCRV”组。
本发明采用常规测试方法表征纳米二氧化硅对正常细胞的相容性以及病毒的抑制能力，首先配置浓度为100μg/mL的nSiO 2培养基溶液，稀释成不同的终浓度（0.75、1.5、3和6μg/mL）处理细胞48 h，发现对CIK细胞毒性很低；再将不同终浓度的nSiO 2培养基溶液与病毒混合后再与细胞孵育，将病毒吸出，继续培养48 h，离心收细胞沉淀，收集的细胞沉淀，一部分利用RNA提取试剂盒提取细胞中的总RNA,另一部分利用蛋白质提取试剂盒提取总蛋白，并用NanoDrop 2000微量紫外分光光度计进行RNA和蛋白质定量，取20μg的蛋白质进行SDS-PAGE分离，并用western blotting对病毒结构蛋白VP7的表达水平进行检测，取2 μg的RNA进行反转录，并用real-time PCR对病毒非结构蛋白基因NS4的表达水平，可以看出纳米二氧化硅对VP7、NS4具有抑制作用；最后比较SiO 2的不同处理方式对GCRV病毒的影响，在三种方式都具有好的效果的基础上，将纳米二氧化硅与病毒先混合再与细胞孵育的方式最佳。因此，根据本发明的启示，可在带有病毒的环境中，喷洒纳米二氧化硅，从而降低病毒对细胞的侵袭。

Claims (10)

  1. 纳米颗粒作为抗病毒纳米材料的应用，或者纳米颗粒在制备抗病毒试剂中的应用；所述纳米颗粒为纳米二氧化硅。
  2. 根据权利要求1所述的应用，其特征在于，所述病毒为DNA病毒、RNA病毒或者朊病毒。
  3. 根据权利要求2所述的应用，其特征在于，所述病毒为呼肠孤病毒。
  4. 根据权利要求1所述的应用，其特征在于，所述纳米二氧化硅以纳米二氧化硅溶液形式存在，纳米二氧化硅溶液的浓度为0.5～10μg/mL。
  5. 根据权利要求1所述的应用，其特征在于，所述纳米二氧化硅的粒径为1～100nm。
  6. 一种利用纳米颗粒提高细胞抗病毒能力的方法，其特征在于，将纳米颗粒与细胞孵育，提高细胞抗病毒能力；或者将纳米颗粒与病毒感染后的细胞孵育，提高细胞抗病毒能力；纳米颗粒为纳米二氧化硅。
  7. 根据权利要求6所述利用纳米颗粒提高细胞抗病毒能力的方法，其特征在于，所述病毒为DNA病毒、RNA病毒或者朊病毒；所述纳米二氧化硅的粒径为1～100nm。
  8. 根据权利要求7所述利用纳米颗粒提高细胞抗病毒能力的方法，其特征在于，所述病毒为呼肠孤病毒；所述纳米二氧化硅的粒径为5～60nm。
  9. 一种利用纳米颗粒降低病毒感染细胞能力的方法，其特征在于，将纳米颗粒与病毒混合，降低病毒感染细胞的能力；纳米颗粒为纳米二氧化硅。
  10. 根据权利要求9所述利用纳米颗粒降低病毒感染细胞能力的方法，其特征在于，将纳米二氧化硅溶液与病毒混合，降低病毒感染细胞的能力；纳米二氧化硅溶液的浓度为0.5～10μg/mL。
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