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

doi:10.21203/rs.3.rs-52066/v1

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

https://doi.org/10.21203/rs.3.rs-52066/v1

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posted 04 Aug, 2020

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A metal nanoparticle composite 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 can inhibit six major clades of 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 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 Tamiflu-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.

Nanoscience

Virology

Nanoparticles

Composite

SARS-CoV-2

COVID-19

Influenza

Virus

The ongoing COVID-19 pandemic has imposed on tremendous threat to humans and global socioeconomics. Many researchers have exerted great efforts to develop effective vaccines and drugs against SARS-CoV-2, the causative virus of COVID-19. Re-purposing of the existing broad-spectrum antiviral agents for COVID-19 therapeutics ¹^,2, including remdesivir, favipiravir, lopinavir and hydroxychloroquine, has been reported to inhibit SARS-CoV-2 based on the in vitro assays. Remdesivir is even recently applied in compassionate use for patients with severe COVID-19 infection ³. In contrast to the small molecules, we consider using metal nanoparticles as the prophylactic of COVID-19 infection. Silver nanoparticle (Ag-NP) and zinc oxide nanoparticle (ZnO-NP) are well-known antimicrobial agents against many kinds of bacteria, fungi and viruses ⁴^,5. Gold nanoparticle (Au-NP) also shows potential use in suppressing human immunodeficiency virus and Mycobacterium tuberculosis bacteria ⁶.

In this study, we formulated a metal nanoparticle composite TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO₂ (42.5 ppm) in aqueous solution with a positive zeta potential of +32.81 mV. The individual metal nanoparticles were synthesized according to our patented method ⁷. In brief, a metal aqueous solution (HAuCl₄, AgNO₃ or ZnCl₂) was reduced by heating with citric acid at 150 ^oC for 12 min, and then dispersed in an appropriate medium to obtain the colloidal metal nanoparticles. The physicochemical properties of the synthesized nanoparticles were fully characterized (Figure S1) by ultraviolet-visible spectroscopy, infrared spectroscopy, inductively coupled plasma atomic emission spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS) analysis and potentiometric titration. According to the TEM imaging, Au-NP, Ag-NP and ZnO-NP are in spherical shape with 20–40, 10–40 and 25–35 nm diameters, respectively. The average sizes of colloidal Au-NP, Ag-NP and ZnO-NP are 78.1, 50.4 and 619.1 nm as shown by the DLS analysis, respectively.

The antiviral activity of TPNT1 against SARS-CoV-2 was first examined by plaque reduction assay. Briefly, Vero E6 cells were infected with SAR-CoV-2 (BetaCoV/Taiwan/NTU01/2020) in the presence of various concentrations of the TPNT1. As shown in Figure 1A, an obvious reduction of plaque numbers and sizes was observed in the presence of 100-fold diluted TPNT1. The calculated IC₅₀ for TPNT1 is 143±15.5-fold dilution (containing 0.71±0.08 ppm Au-NP, 0.71±0.08 ppm Ag-NP, 1.77±0.2 ppm ZnO-NP, and 1.2±0.14 ppm ClO₂). Yield reduction assay was subsequently performed to determine the antiviral activity of TPNT1 by either pretreating the virus with TPNT1 (pretreat + infection), adding TPNT1 during (infection) or after virus infection (post-infection). The schematic illustration for experimental design was shown in Figure 1B. Twenty-four hours after infection, cytopathic effects (CPE) was observed in virus infected cells without pretreatment of virus with TPNT1, and in the infection and post-infection group with or without TPNT1 treatment (Figure S2A). The culture supernatants and cell lysates were harvested for subsequent analysis. The virus copy number in the culture supernatant was determined by quantitative real-time RT-PCR (qRT-PCR) (Figure 1C). The replication of virus in the infected cells was determined by quantification of intracellular viral mRNA and viral nucleocapsid protein (NP) expression using the qRT-PCR of oligo-dT amplified cDNA (Figure 1D), western blot analysis (Figure 1E), and immunofluorescence assay (Figure 1F). Based on the experimental results, pre-incubation of diluted TPNT1 with SARS-CoV-2 is required for efficient inhibition of the virus replication. TPNT1 functioned at a stage before virus infection since the virus titers in the supernatants reduced significantly when TPNT1 was preincubated with virus before infection (Figure 1C). Corresponding reduction of intracellular viral RNA and viral NP protein expression was only observed in cells infected with TPNT1-pretreated viruses (Figure 1D–F). No cell toxicity was observed based on the acid phosphatase (ACP) assay (Figure S2B). The selectivity index (CC₅₀/IC₅₀) was greater than 10.

The ability of TPNT1 to inhibit binding of angiotensin-converting enzyme 2 (ACE2) to SARS- CoV-2 receptor was also confirmed by the ELISA assay using ACE2-Fc-Biotin and spike protein. As shown in Figure 2A, TPNT1 can block binding of spike protein to ACE2-Fc-Biotin in a dose- dependent manner. Furthermore, such inhibition was shown to interfere with the syncytium formation between 293T/Spike/EGFP and H1975-ACE2 cells (Figure 2B) under an inverted fluorescence microscope. A significant inhibition of syncytium formation was demonstrated in the presence of TPNT1 as compared to the solvent (H₂O) control (Figure 2C). Finally, the ability of TPNT1 to inhibit various SARS-CoV-2 was demonstrated using 6 clinical isolates representing 6 major clades of SARS-CoV-2 viruses. As shown in Figure S3, viral RNA in the culture supernatants and cell extracts was significantly inhibited in the presence of TPNT1.

In addition to SARS-CoV-2, we also tested the activities of TPNT1 against influenza virus infection in cell-based assays. Influenza virus infects millions of people annually and can cause severe diseases, especially in elderly ⁸. Although there are several drugs to treat flu clinically, drug-resistant viruses have quickly emerged and thus new therapeutics targeting drug-resistant viruses are needed ⁹. We therefore tested the antiviral activities of TNPT1 against influenza virus. The results showed that TNPT1 effectively relieved the cells with virus-induced toxicity (Figure 3). The effects of TNPT1 did decrease slightly when the cells were infected with extremely high viral loads. Nonetheless, the protection of TNPT1 on the CPE of infected cells is remarkable. In addition, the activities of TNPT1 were detected in the cells infected with wild-type drug- sensitive strains (Figure 3A and 3C) as well as the drug-resistant strains (Figure 3B and 3D) that have H274Y mutation accounting for resistance towards oseltamivir (Tamiflu^TM).

Receptor binding is critical for viral entry and subsequent virus replication in host cells. SARS-CoV-2 uses its spike protein to interact with the ACE2 receptor on host cells to facilitate virus entry and infection ¹⁰^,11. The hemagglutinin (HA) of avian and human influenza viruses is responsible to bind the 2,3-linked or 2,6-linked Neu5Ac receptor on host cells ¹². The SARS- CoV-2 and influenza virions having average diameters of 80–120 nm are in the size range of nanoparticles. As TPNT1 is most effective by pre-incubation with viruses, one possible mechanism for the antiviral activity of TPNT1 may be attributable to bindings of virus surface glycoproteins with the metal nanoparticles, and thus preventing the virions from attachment to host cells ¹³^,14. Indeed, our experiments (Figure 2) support that TPNT1 can block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to ACE2 receptor and to interfere with the syncytium formation. It is suggested that nanoparticles can interact with the sulfur-bearing residues on the viral surface. For example, SARS-CoV-2 spike has 40 cysteine residues while influenza hemagglutinin has 15 cysteine residues. Those residues can easily bind to the metal nanoparticles and the virus entry through these surface proteins would be diminished ¹⁵. Moreover, TPNT1 has positive surface charge (+32.81 mV) that may also enhance the binding with virions ¹⁶.

Inclusion of ClO₂ as an oxidizing agent in TPNT1 nanometal composite is beneficial for virus inhibition. A previous report ¹⁷ indicates that magnesium oxide nanoparticle (MgO-NP) impregnated with Cl₂ exhibits higher bactericidal activity than free Cl₂ or MgO-NP itself. Another study ¹⁸ shows that the combination of single-wall carbon nanotubes and NaOCl (or H₂O₂) also displays a synergistic sporicidal effect. We assume that the ClO₂ component in TPNT1 may render oxidative damage of virus surface, and thus provide enhanced effect for viral inhibition by the metal nanoparticles.

Although metal nanoparticles have been demonstrated to have a wide range of biomedical applications ¹⁹, their toxicity is an issue of concern. The degree of toxicity varies by the type, shape, size, purity, concentration, administration method and exposure time of metal nanoparticles. The current available data from many research teams are insufficient, and some are even contradictory, to determine the adverse effects of metal nanoparticles on human health ^20,21. In this study, all the Au-NP, Ag-NP and ZnO-NP in TPNT1 are prepared in spherical form. In general, spherical metal nanoparticles are non-toxic or less toxic than that in other shapes ²². The amounts of Au-NP (< 1 ppm), Ag-NP (< 1 ppm) and ZnO-NP (< 2 ppm) in the effective dose of TPNT1 tests are within the range of concentration to be used as food additives. The content of ClO₂ (< 1.5 ppm) is also within the safety concentration in drinking water ²³.

Both COVID-19 and influenza are severe infectious diseases. In this study, we have shown that TPNT1 is effective in inhibition of SARS-CoV-2 and influenza viruses, including avian H5N1, human H1N1 and the oseltamivir-resistant H274Y strain. Compared with organic drugs, TPNT1 based on inorganic metal nanoparticles will have low tendency to induce resistant viruses. TPNT1 can also provide prophylactic effects against SARS-CoV-2 and opportunistic infections which are frequently observed in patients suffering SARS-CoV-2 infection by oral gargling, nasal spray, nebulized inhalation or even systemic use after an appropriate clinical trial.

Characterization of nanoparticles

All the reagents were reagent grade and used as purchase without further purification. Tetrachloroauric acid (HAuCl₄, 0.2 M aqueous solution) and zinc powder were purchased from Acros Organics (New Jersey, USA). Silver nitrate (AgNO₃, 0.1 M aqueous solution) was purchased from Merck & Co. (New Jersey, USA). Ultra-pure water was purchased from Hao Feng Biotech Co. (Taipei, Taiwan). Particle size distribution and zeta potential were measured on Otsuka ELSZ-2000ZS dynamic light scattering detector.

Antiviral activities against SARS-CoV-2

The clinical isolate of SARS-CoV-2 was named as SARS-CoV-2/NTU01/TWN/human/2020 (PubMed accession MT066175). Plaque reduction assay was performed in triplicate in 24-well tissue culture plates. SARS-CoV-2 was incubated with TPNT1 for 1 h at 37 °C before adding to the cell monolayer for another one hour. Subsequently, virus-TPNT1 mixtures were removed and the cell monolayer was washed once with PBS before covering with media containing 1% methylcellulose for 5–7 days. The cells were fixed with 10% formaldehyde overnight. After removal of overlay media, the cells were stained with 0.7% crystal violet and the plaques were counted.

ACE2-Fc-Biotin and spike binding by ELISA assay

Fifty microliter of 50 ng/mL purified 1-674 spike proteins were pre-coated on to the 96- well ELISA plate at 4 °C overnight. The plate was first washed three times with PBST (PBS containing 0.05% Tween-20) and blocked with blocking buffer (1% BSA, 0.05% NaN₃ and 5% sucrose in PBS) at room temperature for 30 min. After that, the plate was washed three times with PBST. Serially diluted ACE2-Fc-Biotin with or without TPNT1 were pre-incubated at 37°C for 1 h. After that, the mixture was added to the 96-well plate and incubated at 37 °C for 1 h. After that, the plate was washed three time with PBST and incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:500) at 37 °C for 30 min. After three-times wash with PBST, tetramethylbenzidine substrate (TMB) (T8665, Sigma) was added for 30 min before stopping the reaction by 50 µ L of 1 N H₂SO₄. HRP activity was measured at 450 nm using ELISA plate reader (VERSAMAX).

Syncytia formation

HEK293T cells were co-transfected with plasmid 5 µg pCR3.1-Spike and 0.5 µg pLKO AS2-GFP by lipofetamine 3000 (ThermoFisher, L3000015) for three days before use as effector cells (293T-S). H1975 lung adenocarcinoma cells were transduced with lentivirus encoding full length ACE2 before use as target cells (H1975-ACE2). H1975 and H1975-ACE2 were (2 × 10⁵) cultured in RPMI containing 10% FBS at 37 ^oC for 24 h. 293T-S cells were detached with 0.48 mM EDTA for 5 min. 293T-S (1 × 10⁵) were incubated with H₂O or TPNT1 at 37 ^oC for 1 h. After that, the mixture of TPNT1 and effector cells was added to target cells and incubated at 37 ^oC for 24 h. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min. The 293T/Spike/EGFP cells fused or unfused with H1975-ACE2 cells were counted under an inverted fluorescence microscope (Leica DMI 6000B fluorescence microscope). The percent inhibition of syncytia formation was calculated using the following formula: (100 – (H – L)/(E – L) × 100). H represents the total green fluorescent score in the individual L represents the green fluorescent score in the negative control group with H1975-ACE2 replaced by H1975 as the target cells. E represents the green fluorescent score in each picture in TPNT1 group.

Determination of anti-influenza activities

Recombinant virus Influenza A/WSN/1933 (H1N1), Influenza A/WSN/1933 (H1N1) (NA H274Y), Influenza NIBRG14 (H5N1) harboring the hemagglutinin and neuraminidase from A/Viet Nam/1194/2004 (H5N1) (NA wt), Influenza NIBRG14 (H5N1) (NA H274Y) were produced by using plasmids provided by Dr. King-Song Jeng (Institute of Molecular Biology, Academia Sinica). Influenza virus at indicated titers was mixed with TPNT1 at various dilutions for 1 h at room temperature. The mixtures were used to infect MDCK cells at 1 × 10⁵ cells/mL in 96 wells. After 48 h of incubation at 37 °C under 5% CO₂, the cytopathic effects were determined with CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay reagent (Promega).

Acknowledgements

We would like to acknowledge the service provided by the Biosafety Level-3 Laboratory of the First Core Laboratory, National Taiwan University College of Medicine.

Funding

This research was supported by Academia Sinica [AS-SUMMIT-108] and Ministry of Science and Technology [MOST 107-0210-01-19-01, MOST 108-3114-Y-001-002, MOST107-2320-B- 002-016-MY3].

Author contributions

SYC, , JMF, and PCY are responsible for experimental design and manuscript writing. KYH, TLC, HCK, YHP, and TJRC conducted most of the experiments. LL, CLC and HCH prepared and characterized TPNT1 for testing.

Transparency declarations

The authors declare no competing financial interest.

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SFig1.tif
Figure S1. TEM images, UV-vis spectra and DLS analyses of Au-NP (A), Ag-NP (B) and ZnO-NP (C). Infrared (IR) spectra were recorded on Agilent Technologies Cary630 Fourier transform FT-IR spectrometer. Ultraviolet-visible (UV-vis) spectra were measured on Agilent Technologies Cary60 UV-Vis spectrophotometer. Transmission electron microscopy (TEM) images were recorded on Hitachi H-7100 microscope. Inductively coupled plasma optical emission spectrometry (ICP-OES) was performed on Perkin Elmer Optima 8X00 spectrometer. Particle size distribution and zeta potential were measured on Otsuka ELSZ-2000ZS dynamic light scattering detector.
SFig2.tif
Figure S2. Schematic illustration of SARS-CoV-2 inhibition experiments with TPNT1 and the cytopathic effects (CPE). 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. The CPE induced by SARS-CoV-2 infection of Vero E6 cells in the pretreat + infection, infection-only, and post-infection experiments. Scale bars: 100 µm. C. Viability of Vero-E6 cells on treatment with TPNT1. The clinical isolate used in these experiments was SARS-CoV-2/NTU01/TWN/human/2020 (Accession ID EPI_ISL_408489).
SFig3.tif
Figure S3. Inhibition of various SARS-CoV-2 variants with TPNT1. Yield reduction assay was conducted to determine the antiviral activities of TPNT1 against various SARS-CoV-2 viruses from 6 major clades. Briefly, the virus (multiplicity of infection, M.O.I. = 0.01) was pretreated with TPNT1 or ddH2O (solvent control), and was added to the Vero E6 cells for another one hour. After virus infection, the cells were washed once with PBS, and the cells were maintained in medium without TPNT1 for 24 h. The infected cells were harvested and the amount of SARS-CoV-2 virus RNA in the (A) supernatants and (B) cell extracts was determined by qRT-PCR of E gene using the iTaq™ Univeral Probes One-Step RT-PCR Kit (172-5140, Bio-Rad, USA) and the Applied Biosystems 7500 Real-Time PCR software (version 7500SDS v1.5.1). Plasmid containing partial E fragment was used as the standards to calculate the amount of viral load (copies/uL). NTU03, NTU06, NTU14, NTU16, NTU18, and NTU27 represent B.1, B.2.2, B.1.1, B.1.5, A.1, and B clades of SARS-CoV-2, respectively.

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

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