Respiratory pathogens kill more people than any other infectious agent each year worldwide.1,2 The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection3 has generated a catastrophic scenario both economically and socially, with 6.5 million deaths and 600 million infected people worldwide.4 Development of strategies such as lockdown, face masks, and vaccination have mitigated this problem.5 However, the emergence of new variants with improved transmissibility6 is currently causing an increase in infections and hospitalizations. Airborne transmission of the virus among humans occurs primarily through respiratory aerosols.7 In one scenario, infectious droplets can enter a person's respiratory system when they are close (within one meter) to an infected person. Alternatively, droplet transfer can occur indirectly, where infectious droplets adhere to the surface of an object and are then transmitted to a healthy person who touches the same object.8-9
The use of masks has been a key element to contain the spread of COVID-19, reducing the viral load.10-11 However, it is still necessary to have novel challenges approaches in transmission reduction.12-13 Virucidal coatings could be applied to the surface of the mask, improving both protection and durability. There is considerable interest in harnessing metals, whose ions can inactivate microbes and viruses in minutes on frequently touched surfaces in hospitals and public places.14 Historically, metallic nanoparticles have been shown to display a wide range of virucidal and bactericidal activities due to their efficacy at low doses, small size, high surface area, and acting as ion reservoirs controlling the release of bioactive ions. Metallic nanoparticles demonstrate the ability to generate reactive oxygen species (ROS) to destroy the virus and adhere to DNA or RNA and, consequently, preventing the replication of microorganisms. Since past decades, various types of metallic nanoparticles, such as silver, copper, iron, iron oxide, gold and titanium oxide, with antibacterial and virucidal activity, have been widely described in the literature.15 In this term, copper represent an interesting example in destroying microbial and viral pathogens, more efficiency than zinc and less toxicity than silver.16,17 However, most of the commercial materials -for example in masks- containing copper has focused on the direct use of metal threads or mixtures of copper species at bulk or micromolecular structure. This has the problem of loss of material in each use in addition to presenting a lower efficiency. In addition, most of the currently developed or commercial compounds have been described fundamentally with antibacterial activity and only few and moderate values against COVID-19. 18,19,20
In this work large-scale production of a highly-efficient antiviral nanostructured material composed of very small crystalline copper nanoparticles of a single species, synthesized based on a new biohybrid technology that employs the use of a biological agent for its formation,21-23 has been performed. In addition, this new material has been demonstrated to be excellent stable coating agent on different surfaces of different compositions (cotton, polyester, cellulose, paint, etc.) maintaining 100% virucidal efficiency.
Synthesis and characterization of NanoCu
The copper nanostructured biohybrid material (NanoCu) was prepared by a bio-induced process in the presence of an enzyme (C. antarctica lipase, CALB) at room temperature and aqueous media to grow single crystalline copper phosphate nanoflowers (NFs) as macromolecular structure (Fig. S1). The whole synthetic process (Fig. 1a, see details in Methods) involves three main steps: i) self-assembly interaction between copper and phosphate and coordination between the metal ions and amino acid residues of the protein, ii) coalescence of the Cu nanoparticles and iii) growing of the final macromolecular structure. Scanning electron microscopy (SEM) confirmed the formation of structures of around 1000 ± 3 nm average diameter size (Fig. 1b, see details in Methods). The high-resolution transmission electron microscopy (HRTEM) was employed to confirm that the solid material was constituted by crystalline well-dispersed small copper nanoparticles of average diameter size of around 4 nm (Fig. 1c and Fig. S2).
In order to determine the metal species from which the nanostructured copper hybrid was formed, different X-ray experiments were performed. The wide-angle X-Ray diffraction (XRD) pattern displayed key characteristic peaks of copper orthophosphate, Cu3(PO4)2 (matched well with JCPDS card no. 01-080-0991)24 (Fig. 1d). X-ray photoelectron spectroscopy (XPS) of NanoCu was also performed in order to demonstrate that copper phosphate was the unique species (Fig. 1e, Fig. S3). Analysis by FT-IR also showed the characteristic peak as main one of phosphate at 1048 cm− 1 and 985 cm− 1 (Fig. 1f). Near-circular dichroism (CD) and fluorescence were determined to evaluate the formation of the bioconjugate protein-metal (Fig. 1g-1h). The CD signal showed that the enzyme tertiary structure was clearly altered after copper coordination (Fig. 1g). Figure 1h showed that tryptophan fluorescence spectrum of the enzyme, with a peak centred at 320 nm, was shifted in NanoCu, with a unique clear signal at 385 nm, which is characteristic of a Cu2+-enzyme complex formation.25 Finally, a decrease in the context of α-helix secondary structure, as determined by Far-CD, confirmed the protein-cooper coordination (Fig. 1h). NanoCu stability was evaluated at different conditions, high temperatures, or the presence of different additives, mainly with disinfectant properties, being stable in all cases (Supplementary information details, Fig. S4-5).
In vitro evaluation of antiviral activity against SARS-CoV-2 functional proteins
To determine the antiviral activity of NanoCu against the SARS-CoV-2 virus, first its action against the chymotrypsin-like cysteine protease or main protease (3CLpro or Mpro), involved in virus replication within the human host cell (Fig. S6), was performed.26 Inhibition tests were performed by continuously measuring FRET substrate hydrolysis in the presence of increasing concentrations of NanoCu (Fig. 2a). Complete inhibition of 0.2 µM protease activity were observed even using 10 µg/mL of NanoCu (Fig. 2a-b, Fig. S7). The IC50 value was 0.03 µM Cu, whereas full inhibition was obtained with 0.1 µM Cu (Fig. 2c). The inhibition activity of NanoCu one year after the synthesis were the same (data not shown).
Secondly, Spike (S) protein, in the surface of the virus and which plays a key role in the receptor recognition and cell membrane fusion process, interaction with the angiotensin-converting enzyme 2 (ACE2) protein (Fig. 2d, Fig. S6)27 was evaluated. To assess whether NanoCu interferes with the ACE2-Spike interaction, an ELISA as depicted in Fig. 2d was performed. NanoCu reduced the ability of Spike to interact with ACE2 decreasing the binding in 90% with only 400 µg/mL of material (Fig. 2e). Indeed, this Cu(II) material showed better efficacy than a typical biohybrid synthesized containing Cu(I) nanoparticles (Fig. S8-9).
These results suggested a very different effect of NanoCu depending on the target viral protein. In both cases the action mechanism seemed to be an oxidation process catalyzed by Cu nanoparticles with a different target. In 3CLpro target oxidation mechanism involved the direct oxidation of typical amino acid residues in the protein structure, which has been clearly described by Cu catalysis, such as surface tyrosines and cysteines, for example Cys145 in the active site (Fig. S10). 28
However, a more complex system seems to be occurring in the Spike protein, a glycosylated molecule, where oxidation processes can take place not only in amino acids but also in the glycosylated part (Fig. S11).29–30
Therefore, to determine if the oxidation process was performed in the RBD of spike (Fig. 2f), an RBD construct was treated with NanoCu. The mixture was incubated for different times and then, NanoCu was removed and the RBD structure was evaluated by circular dichroism and fluorescence (Fig. S12). Regarding the CD experiments, the secondary structure of the RBD barely changed after NanoCu treatment, nevertheless there’s a change in the tertiary structure, seen in the near-CD spectra. The tryptophan fluorescence spectral changes were in agreement with time-dependent changes in the environment of aromatic residues is (Fig. 2g-h).
Considering a Fenton-like process where copper with hydrogen peroxide generates the production of hydroxyl radicals, 0.5% (v/v) of hydrogen peroxide was added to the NanoCu emulsion in water. This improved the in vitro inhibition activity of NanoCu against 3CLPro, with a complete inhibition using just 40 ng/mL of product (Fig. 2c), and against RBD-ACE2 binding experiment.
Virucidal properties of NanoCu
Considering the results obtained in the in vitro protein assays, virucidal activity of NanoCu was evaluated in viruses (Fig. 3). To analyze the ability of NanoCu to reduce the viral infectivity, a suspension of NanoCu was incubated with the viruses by rotary shaking at room temperature to determine virucidal activity by plaque assays as standard method to evaluate viral titer of infectious particles (PFU) (Fig. 3a). Firstly, we evaluated the virucidal activity against human coronavirus (HCoV) 229E, a good surrogate of SARS-CoV-2. To accomplish this, different NanoCu concentrations were tested (10, 50, 100, 200 and 500 µg/mL) after an exposure time of 30 and 120 minutes for each one (Fig. 3b and Fig. 3c respectively). The results showed that 200 and 500 µg/mL of NanoCu were enough to reduce the viral viability. We observed a correlation of the virucidal effect produced by NanoCu as a function of time. Subsequently, these two concentrations were validated on SARS-CoV-2 (Fig. 3d,3e). The virucidal effect with 500 µg/mL of NanoCu was confirmed on the SARS-CoV-2 viability. In addition, we evaluated the effectiveness of 200 and 500 µg/mL of NanoCu against a non-enveloped virus, human rhinovirus 14 HRV-14 (Fig. 3f-g), observing a virucidal effect at 30 (Fig. 3f) and 120 minutes (Fig. 3g). In order to see an improvement of the efficiency of the nanomaterial, as previously observed, the virucidal effect of 500 µg/mL of NanoCu against these viruses was studied with the presence of 0.5% (v/v) H2O2 as additive, during 5, 15 or 30 minutes against HCoV-229E (Fig. 3h), SARS-CoV2 (Fig. 3i) and HRV-14 (Fig. 3j). In this case, after 30 minutes of treatment, no plaques forming units (PFU) were detected in any case (reduction of almost 4 log10 (99.99%)). The antiviral efficiency of NanoCu was also evaluated on several bacteriophages (non-human viruses) (Fig. S13). The efficiency of NanoCu was very high against phage f1 and ΦX174, with reduction of almost 4log10 and 6 log10 in 4 h respectively (Figure S13a). In this case also it was demonstrated that efficiency was similar with or without hydrogen peroxide and conserved even when the material had seven-month old showed similar.
NanoCu as antiviral coating agent
First, NanoCu was applied directly as a uniform coating on the surface of a commercial IIR three-layer surgical face mask (cellulose (30%) and polyester (70%)) (Fig. 4a). The material was homogeneously incorporated into the surface of the first layer of a piece of facial mask of 45 cm2 (1/4 of complete mask) by a solution method, where different amounts of NanoCu (125ppm-1000ppm) (Fig. S14-S15) were applied. Scanning electron microscopy (SEM) images of the coated fabric verified the presence of the material adhered to the fibers of the fabric (Fig. 4a. IV), with an optimized amount of 125ppm. This could be attributed to the protein network existing in NanoCu, which can bind to the fabric fiber being embedded on the material and not supported on the surface. Importantly, under these conditions, the amount of material did not obstruct the channels necessary for the breathability of the mask (Fig. 4). Also, the coated mask was treated in the presence of ethanol (from 0 to 100%) and further washed with water at 50ºC and no leaching of NanoCu was observed in any case, confirmed by ICP-MS (data not shown).
NanoCu was subsequently applied to another type of mask, such as FFP2 (Fig. 4b). SEM images show how the material was perfectly adhered to the upper layer of the mask, maintaining breathability (Fig. 4b.IV, Fig. S16). On the other hand, using fabrics as cotton (Fig. 4c) or polystyrene (Fig. S17) similar results were found. Afterwards, the different NanoCu-coated masks were evaluated in terms of potential reuse. For this, two different tests were carried out. First, a piece of IIR mask (45cm2) and a 10.5 cm2 cotton fabric were treated at two temperatures 40ºC and 60ºC for 30, 60 and 120 min (Fig. 4d-4e, Fig. S18). Second, a FFP2 mask was evaluated at 25ºC for six washing cycles (Fig. 4f, Fig. S19). In all cases, between 85% and 100% of NanoCu was maintained on the surface, confirmed by ICP-OES and SEM analyses.
In addition, no traces of Cu were found in the respective second, third and fourth layers inside the mask, remaining exclusively in the outer layer (data not shown).
Other materials such as filter polystyrene or polyester fabrics by using immersion or spraying strategies resulted also in a homogeneous distribution of NanoCu (Figures S20-22).
The catalytic efficiency of NanoCu coated-fabrics was initially tested on the p-aminophenol oxidation assay (see ESI experimental details), yielding in a similar result than using NanoCu in liquid form. Thus, the antiviral efficiency of the NanoCu-coated materials was tested against HCoV-229E and SARS-CoV-2 coronaviruses. The results showed in all cases (cotton, filter and polyester) similar reduction efficiency than those achieved by the material in liquid form, demonstrating the efficiency in dry form which is conserved in surface (Fig. 5a-c). Finally, the virucidal efficiency of the coated-fabrics was tested in HCoV-229E coronavirus inhibition assay after 3 reused previous washes with water or water containing 0.5% H2O2 each cycle, conserving more than 95% efficiency in most cases (Fig. S23).
Finally, applicability of NanoCu as paint coating agent was evaluated. Different concentrations of NanoCu (10%, 20% and 50% (v/v) from 5000 ppm solution) were added to a white paint kindly provided by AC Pinturas (Vélez-Málaga, Spain) (Fig. 6). The paint samples were added to a Petri dish and then dry, (Fig. 6a.1-a.2). SEM analysis of a paint sample with or without NanoCu revealed no changes on the structure (Fig. 6b). TEM analysis of paint containing NanoCu demonstrated the presence of the small copper nanoparticles there (Fig. 6c-d, Fig. S24). NanoCu was extremely stable into the paint at different temperatures (25–70ºC), and for very long times at r.t (Fig. 6e).
Efficiency of the NanoCu in the paint was first confirmed in the pAP assay (Fig. S25A-B). Also, the efficiency of treated paint was confirmed in the 3CLpro assay with full inhibition at 0.4 µg/mL of paints (Fig. S25C).
Then, the virucidal property was demonstrated against HCoV-229E coronavirus (more than 4 log10 (99.99%) was reduced after 30 min incubation) and SARS-CoV-2, even evaluating at different times at 25ºC (Fig. 6f-g). Also, NanoCu in the paint was high efficient against the virus at very low temperature (Fig. 6h-i).
The high stability of NanoCu was also confirmed after washing the painted surface with different commercial disinfectants (Fig. S26).
Optimization, scale up and sustainability footprint of NanoCu
Simple and efficient synthetic procedure are mandatory in a final commercialization of a product. NanoCu synthesis is a green and sustainable process carried out in aqueous media at room temperature without the need for special conditions or special equipment. The synthetic protocol was optimized from 16 h to 1 h and scaled-up from 0.6 L to 10 L in a Syring reactor (Fig. S27). Reproducibility was confirmed after 30 cycles of synthesis where product was identical in all batches, confirmed by XRD (fig. S28), TEM, ICP-OES, and virucidal assay.
The final amount of material added to a complete mask is very low (1–2 mL of 5000 ppm synthesis product, corresponding to 0.0033% of Cu material to total weight of mask (around 3g). This means than 1 L of NanoCu would allow to produce > 10000 antiviral face masks.
Furthermore, to assess its efficiency and sustainability footprint, we compare our copper-coated material with one of the most commercially available which contain copper-containing materials. Cupron® is a well-known commercial material from Cupron company32–33 which is used in the fabrication of textiles, particularly in face mask (2.2% wt. copper). Also, another product is Kuhn all Copper Mask and Kuhn all Copper Insert which are made of 99.95% pure copper mesh that sanitizes and filters the air. The assessment determined in terms of copper amount, virucidal efficiency, final cost, reusability, stability and environment sustainability showed that these technologies exhibited medium or low levels yields mainly because of moderate stability of the material, high price (more than 10€/unit), no reutilization mechanism or even due to very low or negligible virucidal activity at similar copper content. Our technology presented a high yield considering these parameters with high stability product, economically potential available mask, high virucidal efficacy at low copper content, and very environmentally-friendly preparation conditions.