| Repeated outbreaks of infectious diseases caused by enveloped viruses such as influenza A virus(IAV)and severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have posed a great threat to animal and human health.Cold chain logistics become more and more prominent in fresh agricultural products transportation where cold condition benefits the survive of viruses,while the current conventional chemical disinfectants are not effective at low temperature.Therefore,cold chain logistics bring potential risks to the spread of viruses,and it is urgent to develop new disinfectants with antiviral effects at low temperature.Nanozyme is one kind of inorganic nanomaterials with enzyme mimetic catalytic activity.Compared with natural enzymes,traditional nanozymes have lower cost and higher stability,but their enzyme mimetic catalytic activity is still limited.Single-atom nanozymes(SAzymes)are developed by refining the size of the catalytic center to the atomic level in order to improve the catalytic activity of nanozymes.In recent years,SAzymes have been widely used in the field of biomedicine due to high atom utilization efficiency and high catalytic activity.In this study,FeN4P2-single-atom nanozymes(FeN4P2-SAzymes)with low-temperature resistance,high efficiency and broad-spectrum antiviral capacities have been produced,which is of great significance for blocking the transmission of pathogenic microorganisms of major infectious diseases through the cold chain logistics.1.Preparation of FeN4P2-SAzymes and evaluation of its multienzyme mimetic activities at cold chain temperatureIn order to improve the catalytic efficiency of nanozymes,FeN4P2-SAzymes were prepared,and the physical characterization and enzyme mimetic catalytic activity were measured.Electron microscopy showed that FeN4P2-SAzymes had spherical hollow structures with Fe atoms dispersed on the surface.Meanwhile,energy dispersive X-ray spectroscopy showed that FeN4P2-SAzymes only contained Fe,C,N and P,and no other elements were doped.Furthermore,multienzyme mimetic activities showed that FeN4P2-SAzymes had catalytic activities including oxidase-like(OXD-like),peroxidase-like(POD-like)and catalase-like(CAT-like).Importantly,no significant differences were found between low temperatures(4℃ and-20℃)and room temperature(25℃)in enzyme mimetic catalytic activity.These results demonstrated that FeN4P2-SAzymes conformed to the physical characterization of SAzymes and still had multienzyme mimetic catalytic activity at cold chain temperature.2.Evaluation and application of FeN4P2-SAzymes with antienveloped viruses at cold chain temperatureIn order to explore the broad-spectrum anti-enveloped virus properties and applications of FeN4P2-SAzymes,the inactivation effectiveness of FeN4P2-SAzymes against a variety of enveloped and non-enveloped viruses at low temperature,the in vitro and in vivo virulence of FeN4P2-SAzymes-treated IAV,FeN4P2-SAzymes’ application in cold chain outer packaging and personal protective equipment was evaluated.The results showed that FeN4P2-SAzymes could inactivate various enveloped viruses including avian coronavirus(IBV),porcine coronaviruses(PEDV,TGEV,and PDCoV),and other enveloped viruses(multiple subtypes of IAV,NDV,and VSV)in a dose-dependent manner,but had no significant effect on nonenveloped viruses(Adv,PCV-2)at low temperature.Meanwhile,cell apoptosis assay in vitro showed that FeN4P2-SAzymes-treated IAV significantly reduced the apoptosis level of infected cells compared with untreated-IAV group.In vivo pathogenicity test showed that different from single IAV infection group with no mice survival,the survival rate of mice that infected with FeN4P2-SAzymes-treated IAV could reach 100%,and there were no obvious lung lesions or significant increase in lung inflammatory factors(IL-6,IP-10).0.8 mg/cm2 FeN4P2-SAzymes could complete inactivate the viruses on cold chain packaging(-20℃)within 2 h,and inactivate IAV on protective clothing within 15 min at low temperature(4℃).These results indicated that FeN4P2-SAzymes had low-temperature resistance,broad-spectrum resistance to enveloped viruses,and were expected to be used in cold chain outer packaging and personal protective equipment.3.Mechanism of FeN4P2-SAzymes against enveloped virusesIn order to explore the mechanism of FeN4P2-SAzymes against enveloped viruses,this study evaluated the effect of FeN4P2-SAzymes on the lipid peroxidation level of viral lipid envelope and on the structure and function of viral proteins.The results showed that among several natural proteases such as lipoxidase,horseradish peroxidase(HRP)and catalase(CAT),only natural lipoxidase could reduce the virus titer of IAV After FeN4P2-SAzymes were incubated with artificial liposome,FeN4P2-SAzymes significantly increased malondialdehyde(MDA)level compared with untreated group.And compared with untreated virus,FeN4P2SAzymes could increase MDA content in a dose-dependent manner.Further study showed that FeN4P2-SAzymes could degrade on IAV surface proteins,hemagglutinin(HA)and neuraminidase(NA)proteins,and the degradation of HA protein by FeN4P2-SAzymes depended on the presence of liposome.FeN4P2-SAzymes significantly reduced the ability of virus to agglutinate red blood cells and NA activity,and significantly increased the hydroxyl radical(·OH)levels compared with untreated virus.In addition,compared with untreated virus,FeN4P2-SAzymes-treated virus could significantly reduce the invasion ability to cells,thereby affecting the subsequent infection of IAV and resulting in the loss of IAV infection ability.These results indicated that FeN4P2-SAzymes could simulate the OXD-like activity and catalyze the inactivation of a variety of enveloped viruses,including IAV,coronaviruses and so on,mainly by catalyzing lipid oxidation in the lipid envelope membrane of enveloped viruses which could further induce the production of·OH,destroy the structure and function of the membrane adjacent proteins,make the virus lose the infection ability,and eventually lead to virus inactivation. |
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