Nanoparticle composite TPNT1 is effective against SARS-CoV-2 and influenza viruses

Abstract

A metal nanoparticle composite, namely TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO₂ (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 inhibited six major clades of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to the angiotensin-converting enzyme 2 (ACE2) receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and oseltamivir-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.

Figure 1

Transmission electron microscopy (TEM) images, UV–vis spectra and dynamic light scattering (DLS) analyses of metal nanoparticles. (a) Au-NP, (b) Ag-NP, and (c) ZnO-NP.

Figure 2

The antiviral activities of TPNT1 against SARS-CoV-2 in vitro. (a) Inhibition of plaque formation in the presence of serially diluted TPNT1. (b)The half maximal effective concentration (EC₅₀) and cell toxicity of TPNT1 for Vero E6 cells was determined by plaque reduction assay and ACP assay, respectively. (c) Plaque reduction assay of TPNT1(1:50 dilution) against various SARS-CoV-2 strains. (d) The percentage of inhibition by plaque reduction assay in (c). (e) Inhibition of virus titers in the supernatants of SARS-CoV-2 infected H1975-ACE2 cells by TPNT1. (f) Inhibition of viral nucleocapsid (NP) protein expression in TPNT-1 treated SARS-CoV-2 infected H1975-ACE2 cells. The ratio of NP to the internal loading control, proliferating cell nuclear antigen (PCNA), was shown below each blot. At least three independent experiments were performed and one representative result was shown. Full-length blots/gels of (f) were presented in Supplementary Fig. 1.

Figure 3

Inhibition of SARS-CoV-2 entry by TPNT1. (a) Schematic illustration of the (pretreat + infection), infection-only, and post-infection experiments delineates the stage where TPNT1 was added to the viruses or the cells; (b) Quantification of the viral RNA in the supernatants of SARS-CoV-2 infected Vero E6 cells; (c) qRT-PCR detection of viral RNA in the SARS-CoV-2 infected Vero E6 cells ; (d) Western blot of the viral nucleocapsid (NP) protein; and (e) Immunofluorescence assay for the detection of SARS-CoV-2 NP probed with a rabbit monoclonal antibody. Scale bars: 100 µm. At least three independent experiments were performed and one representative result was shown. P < 0.05*; P < 0.001*** Full-length blots/gels of (d) were presented in Supplementary Fig. 2.

Figure 4

Inhibition of SARS-CoV-2 entry by TPNT1. (a) TPNT1 blocks binding of spike proteins to ACE2-Fc-Biotin; (b & c) TPNT1 inhibited syncytium formation between 293 T/Spike/EGFP and H1975-ACE2 cells. At least three independent experiments were performed and one representative result was shown. P < 0.001***.

Figure 5

The antiviral activities of TPNT1 against influenza viruses in vitro. MDCK cells were incubated with (a) WSN (H1N1), (b) oseltamivir-resistant WSN (H1N1)(H274Y), (c) NIBRG14 (H5N1), and (d) oseltamivir-resistant NIBRG14 (H5N1)(H274Y) at various viral inputs in the presence of TPNT1. After 48 h at 35 °C, the virus-induced cytotoxicity was monitored. The cells incubated with virus only were defined as 0% while the cells with no virus inoculation were defined as 100% inhibition.