Virucidal Efficacy of Laser-Generated Copper Nanoparticle Coatings against Model Coronavirus and Herpesvirus

Abstract

High-efficiency antiviral surfaces can be an effective means of fighting viral diseases, such as the recent COVID-19 pandemic. Copper and copper oxides, their nanoparticles (NPs) (CuNPs), and coatings are among the effective antiviral materials having internal and external biocidal effects on viruses. In this work, CuNP colloids were produced via femtosecond laser ablation of the metal target in water, a photophysical, cost-effective green synthesis alternative utilizing sodium citrate surfactant stabilizing the NPs. Raman spectroscopy and X-ray diffraction studies confirmed that the 32 nm mean size CuNPs are mixtures of mainly metallic copper and copper(I) oxide. Polyvinyl butyral was utilized as the binding agent for the CuNPs deposited via high-throughput spray-coating technology. The virucidal efficacy of such coatings containing Cu content ranging from 2.9 to 11.2 atom % was confirmed against animal-origin coronavirus containing ribonucleic acid, the agent of avian infectious bronchitis (IBV), and herpesvirus containing DNA, the agent of bovine herpesvirus (BoHV-1) infection. It was demonstrated that after a short time of exposure, the Cu NP-based coatings do not have a toxic effect on the cell cultures while demonstrating a negative effect on the biological activity of both model viruses that was confirmed by quantification of the viruses via the determination of tissue culture infectious dose (TCID₅₀) virus titer and their viral nucleic acids via determination of threshold cycle (Ct) employing real-time polymerase chain reaction analysis. The assays showed that the decrease in TCID₅₀ virus titer and increase in Ct values correlated with Cu content in Cu NP-based coatings for both investigated viruses. Contact with coatings decreased IBV and BoHV-1 numbers from 99.42% to 100.00% and from 98.65% to 99.96%, respectively. These findings suggest that CuNPs show inhibitory effects leading to the inactivation of viruses and their nuclei regardless of the presence of a viral envelope.

Keywords: copper nanoparticles; coronavirus; herpesvirus; laser ablation in liquid; spray-coating; virucidal surface.

Figure 1

Preparation of Cu NP-loaded coatings and general scheme of the in vitro experiments. (a) Femtosecond laser micromachining setup with a galvanometric scanner and close-up view of the processes in the Petri dish where the Cu target in liquid is scanned with a laser during the ablation process; (b) two-stage spray-coating process starting with deposition of PVB, which is followed by deposition of Cu-NPs, the UV light transmission meter is used for monitoring effective thicknesses of the coatings; (c) cell cytotoxicity test; (d) treatment of virus culture by contacting with investigated coatings and preparation of samples for two quantitative analyses, i.e., virus quantification employing calculating tissue culture infectious dose, and viral nucleic acids via real-time polymerase chain reaction.

Figure 2

Analysis of the Cu colloid synthesized using sodium citrate surfactant. (a) UV–vis–NIR absorbance (optical density, O.D.) spectra of the concentrated colloid. The inset shows a camera image of the concentrated Cu colloid. (b) Raman scattering spectra of the CuNPs with the identified phases addressed in Table S4, “mp”—multiphoton, “a.u.”—arbitrary units. (c) TEM micrograph of the Cu NPs. (d) XRD of the Cu NPs with the indicated hkl indexes for Cu and Cu2O. (e) HRTEM micrograph of Cu NPs with facets addressed to metallic Cu. (f) SAED analysis of the Cu NPs with identified hkl indexes and metallic copper phases.

Figure 3

SEM micrographs of PVB coatings with different Cu NP content, (a) PVB + CuO 10%, (b) PVB + CuO 15%, and (c) PVB + CuO 25% at characteristic magnifications of 1 k×, 20 k×, 100 k× summarized in first, second, and third columns, respectively, with the scale bars of identified length.

Figure 4

In vitro study of different PVB + CuO (10%, 15%, and 25%) coatings and control. (a) Virus titer reduction log10 of IBV, and BoHV-1 after 1 h of contact (*p = 0.0117 and ****p = < 0.0001) and PVB control. (b) Cycle threshold (Ct) values the virus control and coatings of the RT-PCR (*p = 0.0042 and ****p = < 0.0001).