A Copper nanoparticles-based polymeric spray coating: Nanoshield against Sars-Cov-2

Copper nanoparticles (CuNP) synthesis

Three classes of CuNPs differing in diameter sizes were tested and used. Two of these classes, diameters 25 and 60–80 nm, were purchased as being commercially accessible (Io-li-tec, Germany) and generated by laser ablation in liquid. Because the generation of NPs by laser ablation is uncapable to obtain NPs with diameters lower than 25 nm, the third class of CuNPs with diameters of approximately 4–6 nm were synthesized in our laboratory by using CuCl₂·2H₂O (Sinopharm Chemical Reagent Co., Ltd) and L-ascorbic acid (Sinopharm Chemical Reagent Co., Ltd) as previously described. ¹⁰

All the three classes of CuNPs dispersed in water were subsequently characterized by UV-Vis absorption spectra making use of a Jasco V-550 spectrophotometer. In turn, Raman spectroscopy (DXR3xi, Thermo Fisher) was used to further characterize surface-enhanced Raman scattering (SERS) properties of CuNPs.

Coating based on CuNP and polyurethane by spray technology

A medical-grade poly(ether)urethane was dissolved in a mix of Tetrahydrofuran and dioxane a 1:1 (v/v) at a concentration of 0.5% (w/v) (Sigma-Aldrich; St Louis, MO, USA). We added 1% of CuNP after an ultrasonic dispersion of nanoparticles performed at 70°C for 10 min. The solution was sprayed on a rotating mandrel where a filter for face mask has been placed, through a spray gun mounted onto a sliding carriage.

The coating was fabricated using specific distance between the rotating mandrel and the spray guns, mandrel rotating speed, carriage speed, flow rate, and pressure as previously described. ¹¹

At the end of the process, the mandrel with the deposited material was dried at 40°C for 3 h, cut in a circular shape with a diameter of 1.5 cm, placed in a 24 well, and were exposed to UV light in a laminar flow cabinet integrated with a UV light source for 2 h.

SEM analysis

The morphology of the filter no treated, treated with polyurethane (PU) and treated with polyurethane and CuNP (PU-CuNP) was observed using a scanning electron microscope (FlexSEM 1000, Hitachi, Tokyo, Japan). The accelerating voltage was maintained at 5 kV with magnification of 500, 1000, 2000, 3000×. The filter samples were prepared to be conductive by gold coating with a sputter coating unit.

Cells and viruses

African green monkey kidney cells (Vero-E6) were grown as described previously. ¹²

A clinical isolate of SARS-CoV-2, kindly gifted from the San Raffaele Hospital (Milan, Italy) and propagated in Vero-E6 cells, was used for all experiments. All SARS-CoV-2 experiments were performed at a Biosafety Level 3 laboratory.

The propagation of SARS-CoV-2 was performed in Vero-E6 cells as described by Storti et al. ¹²

Virus titration by limited dilution

Viral titer was calculated from the CPE induced through limited dilution of the viral stock. Briefly, 10⁴ Vero-E6 cells were seeded in a 96-well plate and incubated overnight (ON) at standard conditions. The day after, cells were infected in eightfold replicas with 50 µl SARS-CoV-2 at 10 several concentrations obtained by serial 1:10 dilution of the stock (from D0 to D10) and incubated 1 h at 37°C and 5% CO₂.

The Spearman-Karber method has been used to calculate the viral titer using and expressed by Median Tissue Culture Infectious Dose tissue/ml (TCID50/ml). The titer of virus stock was 10⁸ TCID50/ml.

Cytotoxicity assay

The cytotoxic effect of filter coated with polyurethane and CuNP on Vero E6 cells was evaluated using Alamar Blue (Sigma-Aldrich, Milan, Italy). To avoid contamination by bacteria, mold, or other microbes possibly present on metal surfaces, disks were previously soaked in 70% Ethanol at room temperature for 30 min and air dried under a BSL2 cabinet hood. Briefly, pre-cut disks of 14 mm diameter of filter PU and PU-CuNP, were soaked in 200 μl DMEM with no serum for 1 h at room temperature. Two disks per material were examined pooled and added undiluted to a 96-well plate seeded with 5 × 10⁴ Vero-E6/well the day before and cultured in DMEM added with 10% of FBS. Tests were performed using eight replicas for each material. 10 µl/well Alamar Blue were added after 48 h incubation, and plates were incubated for two additional hours. Fluorescence was measured at 590 nm as previously described. ¹³

Antiviral activity assay

A limiting dilution method was carried out with Vero-E6. Briefly, filter disks of 14 mm diameter treated with polyurethane and polyurethane with CuNPs were placed in a 24-well plate. For each experiment four disks were used: two were soaked with the titered virus supernatant, and two with fresh medium. Titered viral supernatant of SARS-COV-2 was diluted in medium with no serum to reach 6 × 10⁴TCID₅₀/ml; 200 μl of this viral suspension or DMEM alone were added to each disk. After incubation at room temperature for 0, 5, 10, 30, and 60 min, viral supernatants and medium were collected, pooled, and used as such or diluted 1:10 up to 1:1000 (dilutions 50–500 μl in DMEM with no serum). Fifty microliter of each sample were added in triplicate to a 96 well plate seeded with 10⁴ Vero-E6 cells/well. Plates were prepared the day before and cells were cultivated in 150 μl DMEM 12.5% serum/well. Positive (e.g. viral supernatant) and negative (medium alone) controls were similarly diluted and added to the plate in triplicate as such or 10-fold diluted. Plates were incubated at 37°C in a 5% humid atmosphere for 4 h after which the medium was replaced with fresh DMEM 10% serum. Plates were incubated for additional 3–4 days until CPE was visible. The presence and extent of CPE were determined by examining the cells with an inverted microscope and the well was scored as virus positive when 75% of cells were lysed. The reduction of infectious viral load was inferred using the Reed and Muench formula. The antiviral activity was assayed in three independent experiments and using two batches of samples.

Fabrication of coating with CuNP and polyurethane

We obtained a coating with 1.5 mg/cm² of CuNP using the spray deposition technique described above. The stability of NP dispersion is the key factor for their application. In this study, we thus used CuNP with a diameter of 25 nm for coating by spray technique because the NP of 60–80 and 4–6 nm showed practical problems with poor dispersion of nanoparticles into the polymer matrix during preparation solution and spray process.

SEM analysis

Figure 1 shows the SEM images from the filter before (no treatment) and after coating with polyurethane alone (PU filter) and with polyurethane-CuNP (PU-CuNP filter) using spray technology. The morphology of the coated filter mask was clearly different from the smooth surface of the untreated filter and showed the CuNPs were uniformly distributed and attached to the rough surface all over the fibers of the filter.

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Figure 1.

SEM images of filter no treated, treated with PU, and treated with PU and CuNP at 500, 1000, 2000, and 3000× magnifications.

Antiviral activity against SARS-CoV-2

Analysis of antiviral activity was preceded by several experiments aimed to determine the cytotoxicity of the filters coated with polyurethane and CuNP. Our data showed that filters do not release harmful substances into the culture medium and have no detrimental effects on the cells.

Antiviral tests were set in such a way to test in parallel the same viral preparation exposed at various times to the filters and then incubated with the cells until the CPE was overtly visible by microscope reading or replaced after 4 h with fresh medium. Since replacement of the medium did not alter significantly viral infectivity we used 3000 TCID₅₀ as viral input dose, an amount that permitted precise evaluation of the antiviral activity of testing material.

The antiviral activity was assayed by the limiting dilution method in three independent experiments and using two batches of filters PU and filter PU-CuNP. Table 1 shows the residual SARS-CoV-2 infectious dose following contact with filter coated with PU and CuNP (PU-CuNP filter) expressed as reduction of TCID₅₀ and log TCID₅₀.

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Table 1.

Residual SARS-CoV-2 infectious dose following contact with filter coated with PU and CuNP.

PU-CuNP filter	Contact time (min)
	0	5	10	30	60
TCID50	3981.0717	316.2278	218.7762	31.6228	0
% TCID50 reduction		92.06	94.50	99.21	100
Log TCID50	3.6	2.5	2.34	1.5	−8
% Log TCID50 reduction		30.56	35	58.33	322.22

CuNP: copper nanoparticle; PU: polyurethane; TCID50: Medium Tissue Culture Infectious Dose 50%/ml.

Filter Cu-NP showed a strong virucidal activity that increased proportionally with the time of contact with the materials. As shown in Table 1, the reduction was noticeable since the first time examined, 5 min, increased three times with only five further minutes of incubation and abated 99% viral infectivity in 30 min. At 1-h contact, the virus was inactivated to a point that CPE was observed in only 1/18 wells. A similar result was obtained with the positive control virus diluted 1:1000 (CPE observed in 3/18 wells). In all, this indicated that Copper abates viral infectivity over three logs in 1 h of contact. Figure 2 shows the time-dependent reduction of viral titer with PU and with PU-CuNP filters. The reduction of SARS-CoV-2 titer after incubation with CuNP was very high in the first 5 min and then slowly decreased as shown by the reduction of the slope (Figure 2).

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Figure 2.

Reduction of viral titer after incubation with filter treated with CuNP and PU. The reduction of the SARS-CoV-2 titer was shown after incubation with a filter treated with CuNP and PU at different time points. After only 5 min of incubation, the effect produced by CuNP is relevant and decreases during the time.