Title：Diatomic iron nanozyme with lipoxidase-like activity for efficient inactivation of enveloped virus

Journal：《Nature Communications》

IF：16.6

Original link：https://www.nature.com/articles/s41467-023-43176-4

Reporter：Xiang Li, Master of Grade 2022

Enveloped viruses encased within a lipid bilayer membrane are highly contagious and can cause many infectious diseases like influenza and COVID-19, thus calling for effective prevention and inactivation strategies. Here, we develop a diatomic iron nanozyme with lipoxidase-like (LOX-like) activity for the inactivation of enveloped virus. The diatomic iron sites can destruct the viral envelope via lipid peroxidation, thus displaying non-specific virucidal property. In contrast, natural LOX exhibits low antiviral performance, manifesting the advantage of nanozyme over the natural enzyme. Theoretical studies suggest that the Fe-O-Fe motif can match well the energy levels of Fe2 minority β-spin d orbitals and pentadiene moiety π* orbitals, and thus significantly lower the activation barrier of cis,cis-1,4-pentadiene moiety in the vesicle membrane. We showcase that the diatomic iron nanozyme can be incorporated into air purifier to disinfect airborne flu virus. The present strategy promises a future application in comprehensive biosecurity control.

Enveloped viruses are composed of external lipid membranes to protect the genetic material in their life cycle. Many infectious diseases are caused by enveloped viruses, such as Ebola virus, influenza virus, Dengue fever virus, Zika virus, and SARS-CoV-21,2,3,4,5,6,7. Taking influenza A viruses (IAVs) for example, they have caused four worldwide influenza pandemics since the last century4,8. Various vaccines and antiviral drugs have been developed for the prevention and treatment of IAVs8,9. However, antigenic shift and drift, drug resistance, and the diversity of influenza subtypes can compromise the effectiveness of these strategies9,10,11. Therefore, it is imperative to develop comprehensive biosecurity control measures such as environmental disinfection and interruption of virus transmission.

1. Synthesis and characterization of Fe₂DAC

Fig.1. The synthesis of Fe₂DAC nanozyme. a) Schematic illustration of the macrocyclic-precursor Fe₂L mediated synthesis of Fe₂DAC. b) TEM image of Fe₂DAC. c) Energy-dispersive X-ray elemental mapping of Fe₂DAC. d) AC HAADF-STEM images of Fe₂DAC, wherein the yellow ellipses mark the metal pairs. 

Fig.2. The characterization of Fe2 DAC nanozyme. XANES (a), FT EXAFS (b), and WT EXAFS (c) spectra of Fe₂DAC, Fe SAC, and other references. d) The FT EXAFS fitting of Fe₂DAC and Fe₂L at the R-space. Inset shows the Fe₂N₄O₂ model that used for data fitting for Fe₂DAC (C: gray; N: blue; O: red; Fe: pale purple). e) The TOF-SIMS spectra of Fe SAC, Fe₂ DAC, and Fe₂L.

2. LOX-like activity and destruction of viral envelope

Fig.3. Enzyme-like activity of Fe₂DAC. a) Kinetics for OXD-like activity of Fe₂DAC, Fe SAC and blank (decarbonized ZIF-8), respectively. b) The level of lipid peroxidation (MDA detection) after liposomes were treated by Fe₂DAC. c) SEM images of liposomes treated by Fe₂DAC (500 µg/mL). d) Schematic illustration of lipid destruction upon treatment with Fe₂DAC.

Fig.4. Fe₂DAC compromises the viral envelope and neighboring proteins of IAVs through lipid peroxidation. a) The level of lipid peroxidation (MDA detection) when H1N1 IAVs were treated by Fe₂DAC. b) TEM image of IAVs treated by Fe₂DAC (500 µg/mL) for 90 min at RT. c) Circular dichroism analysis of the protein structure of IAVs treated by Fe₂DAC for 90 min at RT. d) Western blot analysis of hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) proteins of IAVs treated by Fe₂DAC for 90 min at RT. e) Quantification of the HA, NA and NP protein levels of H1N1 IAVs after treatment with Fe₂DAC. 

3. Non-specific antiviral effect and application in air filter

Fig.5. The nonspecific antiviral effect of Fe₂DAC and application in air filter for the inactivation of airborne flu virus. a) HA titer of Fe₂DAC-treated H1N1 (purified virus) IAVs, under 15/30/60/90 min. b) HA titer of Fe₂DAC-treated H1N1 (purified virus) IAVs at 37 °C and 4 °C, under 90 min. c) HA titer of Fe₂DAC-treated H9N2 (purified virus) IAVs, under 15/30/60/120 min. d) HA titer of H1N1 IAVs treated with Fe₂DAC coated nonwoven HEPA filter, under 90 min. 

    4. Mechanistic insights into the LOX-like activity

Fig.6. Molecular mechanism of LOX-like enzymatic activity. a) The proposed reaction pathway of LOX-like activity, involving the dioxygenation of a substrate containing a cis,cis-1,4-pentadiene moiety on Fe₂N₄O₂ model. Bond distance unit: Å. b) Gibbs free energy profile for key intermediate and transition states in the LOX-like catalytic cycle. Energy unit: eV. c) Electronic structure analysis of projected electronic densities of states (pDOS) of a cis,cis-1,4-pentadiene moiety, Fe₂DAC structure, and their interaction configuration. The asterisk (*) is used to mark species adsorbed on Fe₂DAC.

In summary, we prepared a Fe2 DAC nanozyme through a macrocyclic precursor-mediated encapsulation-pyrolysis approach. The diatomic iron motif was identified by the combined capacities of HAADF-STEM, EXAFS, and TOF-SIMS. We demonstrated the multienzymtic activities of the Fe2 DAC. Particularly, the Fe2 DAC has shown distinctive activity toward lipid peroxidation, which was not observed on the Fe SAC counterpart, suggesting the uniqueness of the neighboring iron sites. DFT calculations suggest that the Fe-O-Fe motif can effectively activate the cis,cis-1,4-pentadiene moiety in lipid by matching the energy levels of the Fe2 minority β-spin d orbitals and pentadiene moiety π* orbitals. The LOX-like activity of Fe2 DAC makes it possible to destruct the envelope of influenza viruses and thus efficiently reduce their HA titer and TCID50 values over a wide temperature range. This strategy has great potential for the inactivation of many other enveloped viruses. Compared with natural LOX, the higher antivirus efficiency of Fe2 DAC may come from its more open active sites. As a proof-of-concept, we showed that the Fe2 DAC can be integrated to air cleaning devices in the form of catalyst coating for the inactivation of airborne viruses, which provides a promising strategy for comprehensive biosecurity control.