FACIAL PROTECTION ELEMENT FOR PROTECTION AGAINST RISKS OF EXPOSURE TO INFECTIOUS BIOLOGICAL AGENTS

Abstract

The present invention describes a facial protection element for protection against risks of exposure to infectious biological agents, such as microorganisms, like a virus with an envelope, including SARS-CoV-2 and the multidrug-resistant Gram-positive bacteria. Specifically, the present invention relates to a facial protection element of the type comprising: face shields, glasses, masks, counter shields, etc., which prevent not only direct exposure to the infectious biological agent, but rather also inactivate same once the biological agent comes into contact with the facial protection element. The facial protection element comprises a plastic material or glass with a biocidal agent coating layer.

Claims

1. A facial protection element with a biocidal effect comprising a body with at least one surface made of transparent and impermeable plastic material or glass, characterized in that said surface comprises a coating layer of a biocidal agent.

2. The facial protection element according to claim 1, characterized in that the biocidal agent coating layer comprises at least one compound selected from: triclosan, citric acid, acetic acid, sodium 2-biphenylate, sodium hypochlorite, salicylic acid, alkyldimethyl benzyl ammonium chloride, hydrogen peroxide, sodium dichloroisocyanurate, didecyldimethylammonium chloride, C12-16 alkyldimethyl benzyl ammonium chloride, sodium dichloroisocyanurate, sodium dichloroisocyanurate dihydrate, propan-1-ol, glutaraldehyde, peracetic acid, isopropyl alcohol, ethanol, benzyl-C12-C16-alkyldimethyl ammonium chloride, pentapotassium bis(peroxymonosulfate) bis(sulfate), C12-18 saccharinate, orthophenylphenol, orthophenylphenol, chlorine dioxide, pentapotassium bis(peroxymonosulfate) bis(sulfate), didecyldimethylammonium chloride, N-dichlorofluoromethylthio-N′,N′-dimethyl-N-phenyl-sulfamide (dichlofluanid), pyridine-2-thiol-1-oxide, sodium salt (sodium pyrithione), sulfur dioxide, sodium bromide, ammonium sulfate, silver, silver chloride, ammonium bromide, potassium 2-biphenylate, bromine chloride, sodium p-Chloro-m-cresolate, mixture of cis- and trans-p-menthane-3,8-diol (citriodiol), tetrakis(hydroxymethyl)phosphonium sulfate (1:2) (THPS), didecyldimethylammonium chloride (DDAC (C.sub.8-10)), 6-(phthalimido)peroxyhexanoic acid (PAP), tetrachlorodecaoxide complex (TCDO), active chlorine: obtained by the reaction of hypochlorous acid and sodium hypochlorite produced in situ, silver and zinc zeolite, silver and copper zeolite, esfenvalerate/(S)-α-cyano(3-phenoxybenzyl) (S)-2-(4-chlorophenyl)-3-methylbutyrate (esfenvalerate), boric acid, borax, copper nanoparticles, gold, silver, graphene, graphene oxide, fullerene, carbon dots, graphite, reduced graphene oxide, reduced graphene, simple or multi-walled carbon nanotubes, carbon nanofibers, fullerene, alkylamine- or ammonia-functionalized graphene oxide, boron doped graphene, nitrogen doped graphene, phosphorus doped graphene, sulfur doped graphene, boron and nitrogen doped graphene, phosphorus and nitrogen doped graphene, sulfur and nitrogen doped graphene and sulfonated reduced graphene oxide, graphene/TiO.sub.2, graphene/Fe.sub.3O.sub.4, graphene/Mn.sub.3O.sub.4, graphene/Pd, graphene/Pt, graphene/PtCo, graphene/PtPd, reduced graphene/TiO.sub.2, reduced graphene/Fe.sub.3O.sub.4, reduced graphene/Mn.sub.3O.sub.4, reduced graphene/Pd, reduced graphene/Pt, reduced graphene/PtCo and reduced graphene/PtPd, geraniol, thymol, citrodiol, sanicitrex, condensed tannins, procyanidin or proanthocyanidins A, B, C and D (A1, A2, A3, A4, B1, B2, B3, C1, C2, C3, D1, D2, D3, etc.), grapeseed extract, cranberry, blueberry, and blackberry extract, cocoa extract, flavanols, flavonoids, afzelechin, cinnamon extract, green and black tea extract, catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, proanthocyanidins, theaflavins, thearubigins, polyphenols, citronella and compounds of quaternary ammonium and derivatives thereof, such as: benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, cetrimide, tetraethylammonium bromide, didecyldimethylammonium chloride, domiphen bromide, benzalkonium chloride.

3. The facial protection element according to claim 2, characterized in that the biocidal agent comprises at least one quaternary ammonium compound selected from: benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, cetrimide, tetraethylammonium bromide, didecyldimethylammonium chloride, domiphen bromide, and benzalkonium chloride.

4. The facial protection element according to claim 1, characterized in that the thickness of the coating is between 0.001 to 100 μm.

5. The facial protection element according to claim 4, characterized in that the thickness of the coating is 25 μm.

6. The facial protection element according to claim 1, characterized in that the plastic material is a thermoplastic material, made from synthetic or natural and/or biodegradable plastic materials as well as mixtures thereof.

7. The facial protection element according to claim 6, characterized in that the plastic material is a thermoplastic material synthetic selected from: polyethylene terephthalate (PET), polyvinyl chloride (PVC), polypropylene (PP), high or low density polyethylene (PE), polycarbonate (PC), polymethyl-methacrylate (PMMA), polystyrene (PS), polyhydroxyethyl acrylate (PHEA), polyhydroxyethyl methacrylate (PHEMA), polyamide or nylon (PA), polybutylene (PB) and Teflon (or polytetrafluoroethylene, PTFE), polypyrrole (PPy), polyaniline, polythiophene, poly(3,4-ethylenedioxythiophene), poly(3,4-ethylenedioxythiophene, poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate), polythiophene-vinylidene), poly(2,5-thiethylene-vinylidene), poly(3-alkylthiophene), poly(p-phenylene), poly-p-phenylene-sulfide, poly(p-phenylenevinylene), poly(p-phenylene-terephthalamide), poly(3-octylthiophene-3-methylthiophene), and poly(p-phenylene-terephthalamide), poly(vinyl alcohol) (PVA), as well as mixtures thereof.

8. The facial protection element according to claim 6, characterized in that the natural and biodegradable material is selected from chitosan, hyaluronic acid, carrageenan, starch, acacia gum, xanthan gum, pectins, proteins, chitin, pullulan, non-crosslinked alginate, calcium alginate, zinc alginate, strontium alginate, and other alginate hydrogels crosslinked with other divalent cations, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxypropionate), poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(3-hydroxyoctanoate), poly(3-hydroxyoctadecanoate), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(5-hydroxyvalerate)gelatin, and/or collagen, as well as mixtures thereof.

9. The facial protection element according to claim 1, characterized in that the biocidal effect comprises inactivation of human coronavirus (HCoV), hepatitis B virus (HVB), hepatitis C virus (HCV), hepatitis D virus (HDV), dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus, Western Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), tick-borne encephalitis virus (TBEV), Rift Valley fever virus (RVFV), herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), human T-lymphotropic virus (HTLV), Epstein Barr virus (EBV), human cytomegalovirus (HCMV), Lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV), Nipah virus, influenza A virus, influenza B virus, influenza C virus, Hantaan virus (HTNV), Junin virus, rabies virus, Ebola virus, Marburg virus (MARV), Zika virus (ZIKV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), human immunodeficiency virus (HIV), varicella-zoster virus (VZV), measles virus, mumps virus (MuV), human respiratory syncytial virus (RSV), rubella virus (RuV), as well as multidrug-resistant Gram-positive bacteria, such as Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Enterococcus faecalis, and Enterococcus faecium, after contact with the surface made of plastic material comprising the coating.

10. The facial protection element according to claim 1, characterized in that it is presented in the form of a face shield, helmet, glasses, mask, space partitioning shield of the type intended for a counter or vehicles.

Description

BRIEF DESCRIPTION OF FIGURES

[0033] FIG. 1 depicts a facial protection shield made of plastic material.

[0034] FIG. 2 depicts AFM topography images (height), amplitude error (E Amp), 2D phase images, and a 3D phase representation, recorded in tapping mode, of the untreated plastic (U Plastic), plastic treated by means of dip-coating with the ethanol-based solvent (S Plastic), and filter with the benzalkonium chloride (BAK) biofunctional coating (BAK Plastic) by scanning a 10 μm×10 μm area.

[0035] FIG. 3 illustrates the morphology of the BAK biofunctional coating on the PET plastic by means of field-emission scanning electron microscopy with EDS for elemental analysis. PET plastic with 0.182±0.034% w/w of BAK biofunctional coating (BAK Plastic) at two magnifications: (a) ×150 and (b) ×720, and (c) EDS for elemental analysis of the coating and the PET matrix.

[0036] FIG. 4 depicts the opacity results of the untreated plastic (U Plastic), plastic treated by means of dip-coating with the ethanol-based solvent (S Plastic), and filter with the BAK biofunctional coating (BAK Plastic). The results of the ANOVA statistical analysis are indicated in this graph; ns: not significant.

[0037] FIG. 5 depicts the antibacterial disk diffusion tests in agar. Untreated plastic (U Plastic), plastic treated by means of dip-coating with the ethanol-based solvent (S Plastic), and plastic treated with the BAK biofunctional coating (BAK Plastic) after 24 hours of culture at 37° C. The normalized distances of the antibacterial halos, expressed as the mean±standard deviation and calculated with equation (1), are shown in each image.

[0038] FIG. 6 depicts the loss of viability of phage phi6 measured by the double-layer method. Phage 6 titration images of undiluted samples for control, untreated plastic (U Plastic), plastic treated with a filter by means of dip-coating with the ethanol-based solvent (S Plastic), and plastic with BAK biofunctional coating (BAK Plastic) at 1 min of viral contact.

[0039] FIG. 7 depicts the reduction of the infectious titers in PFU/mL determined by the double-layer assay for phage phi6. Logarithm of plaque forming units per mL (log (PFU/mL)) of the control, untreated plastic (U Plastic), plastic treated with a filter by means of dip-coating with the ethanol-based solvent (S Plastic), and disk with the BAK biofunctional coating (BAK Plastic) at 1 minute of viral contact.

[0040] FIG. 8 depicts the reduction of infectious titers in PFU/mL of SARS-CoV-2 after 1 min of contact determined by the TCID50/mL method. Untreated plastic (U plastic), plastic treated with the ethanol solvent (S Plastic), disk with the BAK biofunctional coating (BAK Plastic). The set of experimentally measured points are depicted by means of dots, squares, and triangles.

DETAILED DISCLOSURE OF AN EMBODIMENT OF THE INVENTION

[0041] 1. Materials and Methods

1. 1. Dip-Coating with Benzalkonium Chloride Solution

[0042] The facial protection element in the form of treated protective shield treated as follows was acquired from Plsticos Villamarchante S.L (http://plasticosvillamarchante.com/. The transparent plastic forming the protection shield is made of polyethylene terephthalate (PET). The front black piece and the rear black strap are made of polypropylene (PP) (FIG. 1). The transparent PET plastic has a thickness of 0.3167±0.0408 mm. The coating with benzalkonium chloride (BAK) was performed by means of the dip-coating method to achieve a BAK content of 0.182±0.034% w/w on the surface of the PET (mass percentage that remains of BAK on the final PET plastic after arranging the BAK coating).

[0043] Six replicates (n=6) of each type of sample were tested by cutting PET disks from the protective shield measuring about 10 mm in diameter with a cylindrical punch. Six replicates of untreated plastic measuring 10 mm in diameter were also cut in order to be used as reference material. The disks were treated as described by introducing 6 disks in a beaker that contained 100 ml of solution for 30 minutes at 25°, and then they were dried at 60° for 48 hours. The disks were provided treatment with UV radiation one hour per side after the drying as a method of sterilizing the disks.

1.2. Atomic Force Microscopy (AFM)

[0044] Atomic force microscopy (AFM) was performed with a Bruker MultiMode 8 SPM, operating in tapping mode in air and with the NanoScope V controller and NanoScope 8.15 software. A cantilever of antimony doped silicon (n) by Bruker with a scanning speed of 0.500 Hz was used. The phase signal was set to zero at the resonant frequency of the tip. The tapping frequency was 5%-10% less than the resonant frequency. The amplitude of the unit and the amplitude set point were 308.5 and 644.8 mV, respectively, and the aspect ratio was 1.00.

[0045] In the atomic force microscope (AFM), a sharp tip located at the end of a flexible cantilever runs over the surface of a sample, keeping a small interaction force constant. The scanning movement is performed by a piezoelectric scanner, and tip/sample interaction is monitored, reflecting a laser off the rear part cantilever, which is picked up in a photodiode detector. The photodiode is split into 4 segments, and the voltage differences between the different segments (generally the top 2 with respect to the bottom 2) precisely determine the changes in inclination or oscillation amplitude of the tip.

[0046] Tapping AFM: Measures the topography by intermittently touching the surface of the sample with an oscillating tip. Lateral and pressure forces which may damage soft samples and reduce image resolution are eliminated. It can be performed in the air and in liquid medium.

[0047] Phase image: Provides images in which the contrast is caused by differences in the adhesion and viscoelasticity properties of the surface of the sample. It is performed in tapping mode and measured as the delay in the oscillation phase of the tip measured in the photodiode, with respect to the value of oscillation phase provided by the piezo of the support for the tip.

1.3. Electron Microscope

[0048] A Zeiss Ultra 55 field-emission scanning electron microscope (FESEM, Zeiss Ultra 55 model) was operated at an acceleration voltage of 10 kV to observe the morphology of the BAK biofunctional coating on the treated PET surface at two magnifications (×150 and ×720). The plastic samples were prepared to be conductive by means of coating with platinum using a cathode sputtering coating unit. This electron microscope is equipped with energy-dispersive X-ray spectroscopy (EDS) to estimate the elemental ratio at 2.00 kV.

1.4. Transparency

[0049] The transparency or opacity of the treated and untreated samples was evaluated according to the spectrophotometric method used by Park and Zhao [S. II Park, Y. Zhao, Incorporation of a high concentration of mineral or vitamin into chitosan-based films, J. Agric. Food Chem. 52 (2004) 1933-1939. doi: 10.1021/jf034612p]. Therefore, the rectangular samples (4 mm×50 mm) of the treated and untreated samples were placed directly in a spectrophotometer cell to measure absorbance at 600 nm with a UV/VIS Nanocolor UV0245 spectrophotometer (Macherey-Nagel, Germany). The treated and untreated samples were dried at 60° C. for 24 hours before each measurement, and an empty cell was used as a reference. After that, the opacity (O) of the films can be determined using equation (1), where Abs600 is the value of absorbance at 600 nm and x is the thickness of the sample in mm.

O=Abs600/x  (1)

[0050] The measurements were taken with three samples of each material to ensure the reproducibility of the measurements and were calculated as absorbance divided by the thickness of the film (expressed in mean±standard deviation).

1.5. Bacterial Culture for Phage Phi6

[0051] Was cultured Pseudomonas syringae(DSM 21482) from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) in solid tryptic soy agar (TSA, Liofilchem) and then in liquid tryptic soy broth (TSB, Liofilchem). Incubation of the liquid was carried out at 25° C. and 120 rpm.

1.6. Propagation of Phage Phi6

[0052] The propagation of phage phi6 (DSM 21518) was carried out according to the specifications provided by the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany).

1.7. Antiviral Test Using the Biosafe Viral Model

[0053] A 50 μL volume of a phage suspension in TSB was added to each filter at a titer of about 1×10.sup.6 plaque forming units per mL (PFU/mL) and was left to incubate for 1, 10, and 30 min. Each filter was placed in a Falcon tube with 10 mL of TSB and was sonicated for 5 min at 24° C. After that, each tube was stirred with a vortex for 1 min. Serial dilutions of each Falcon were performed for phage titration, and 100 μl of each phage dilution were brought into contact with 100 μl of the host strain at OD 600 nm=0.5. The infectious capacity of the phage was measured based on the double-layer method [Kropinski, A. M.; Mazzocco, A; Waddell, T. E.; Lingohr, E.; Johnson, R. P. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol. Biol. 2009, 501, 69-76], where 4 ml of top agar (TSB+0.75% bacteriological agar, Scharlau) and 5 mM CaCl.sub.2) were added to the mixture of phages and bacteria poured into TSA plates. The plates were incubated for 24 to 48 h in an oven at 25° C. The phage titer of each type of sample was calculated in PFU/mL and compared with the control, i.e., 50 μL of phages added to the bacterium without coming into contact with any filter and without being sonicated. The antiviral activity in log reductions of titers was determined at 1, 10 and 30 min of contact with the viral model. It was checked that the residual amounts of disinfectants in the titrated samples did not interfere with the titration process and that the sonication-vortex treatment did not affect the infectious capacity of the phage. Antiviral tests were performed three times on two different days (n=6) to ensure reproducibility.

1.8. Antiviral Tests with SARS-CoV-2

[0054] The SARS-CoV-2 strain used in this study (SARS-CoV-2/Hu/DP/Kng/19-027) was kindly provided by Dr. Tomohiko Takasaki and Dr. Jun-Ichi Sakuragi of the Public Health Prefectural Institute of Kanagawa. The virus was plaque purified and propagated in Vero cells. SARS-CoV-2 was stored at −80° C. A 50 μL volume of a virus suspension in phosphate-buffered saline (PBS) solution was added to each filter at a titer dose of 1.3*105 TCID50/filter and was then incubated for 1 min at room temperature. Then 1 ml of PBS was added to each filter, and it was then stirred with a vortex for 5 min. After that, each tube was stirred with a vortex for 5 min at room temperature.

[0055] The viral titers were determined by means of mean tissue culture infectious doses (TCID50) in a level 3 biosafety lab at the University of Kyoto. Briefly, TMPRSS2/Vero cells [Matsuyama, S.; Nao, N.; Shirato, K.; Kawase, M.; Saito, S.; Takayama, I.; Nagata, N.; Sekizuka, T.; Katoh, H.; Kato, F.; et al. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc. Natl. Acad. Sci. USA 2020, 117, 7001-7003] (JCRB1818, JCRB Cell Bank), cultured with minimum essential medium (MEM, Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, were seeded in 96-well plates (Thermo Fisher Scientific). The samples were subjected to serial dilution 10 times from 10.sup.−1 to 10.sup.−8 in the culture medium. The dilutions were placed in the TMPRSS2/Vero cells in triplicate and incubated at 37° C. for 96 h. The cytopathic effect was evaluated under a microscope. TCID50/ml was calculated by means of the Reed-Muench method.

1.9. Antibacterial Tests

[0056] Antibacterial disk diffusion tests in agar were performed to analyze the antibacterial activity of the treated and untreated plastics [Marti, M; Frigols, B.; Serrano-Aroca, A. Antimicrobial Characterization of Advanced Materials for Bioengineering Applications. J. Vis. Exp. 2018, e57710.; Shao, W; Liu, H.; Liu, X.; Wang, S.; Wu, J.; Zhang, R.; Min, H.; Huang, M. Development of silver sulfadiazine loaded bacterial cellulose/sodium alginate composite films with enhanced antibacterial properties. Carbohidr. Polym. 2015, 132, 351-358.]. The methicillin-resistant bacterium Staphylococcus aureus, COL [Gill, S. R.; Fouts, D. E.; Archer, G. L.; Mongodin, E. F.; DeBoy, R. T.; Ravel, J.; Paulsen, I. T.; Kolonay, J. F.; Brinkac, L.; Beanan, M.; et al. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J. Bacteriol. 2005, 187, 2426 2438.], and methicillin-resistant Staphylococcus epidermidis, RP62A [Christensen, G. D.; Bisno, A. L.; Parisi, J. T.; McLaughlin, B.; Hester, M. G.; Luther, R. W. Nosocomial septicemia due to multiple antibiotic-resistant Staphylococcus epidermidis. Ann. Intern. Med. 1982, 96, 1-10.] were used at a concentration of about 1.5*10.sup.8 CFU/ml in tryptic soy broth, cultured in trypticase soy agar plates. The sterilized disks were placed on the bacterial lawn to incubate them aerobically at 37° C. for 24 h. The antibacterial activity of the assayed filter disks was expressed according to equation (1) [Marti, M.; Frigols, B.; Serrano-Aroca, Á. Characterization of Advanced Materials for Bioengineering Applications. J. Vis. Exp. 2018, e57710.]:

1.10. Statistical Analysis

[0057] The statistical analyses were performed by means of ANOVA followed by Tukey's test for post hoc analysis (*p>0.05, ***p>0.001) in GraphPad Prism 6 software (gGraphPad Software Inc., San Diego, Calif., USA).

[0058] 2. Results

2.1. Coating Morphology

[0059] Atomic force microscopy, electron microscopy, and elemental analysis were performed to characterize the BAK microcoating formed on the PET plastic. FIG. 2 shows the AFM images of the treated and untreated PET plastic on a scanning area measuring 10 m×10 m. The images of the topography and the phase angle clearly indicate that a BAK coating is formed on the surface of the PET after the dip-coating treatment with the solvent containing BAK (FIG. 2). FIG. 2 shows that the untreated plastic (U Plastic) presents some impurities on its surface which disappear after immersing the disk in 70% ethanol for 30 minutes. Therefore, surface imperfections produced by the plastic manufacturing method can clearly be observed in the 2D phase image for S Plastic. However, BAK plastic AFM images clearly show that a BAK coating covering all the imperfections observed in the 2D phase image of the S Plastic sample was formed, according to the FESEM images shown in the following FIG. 3. This coating has slightly higher areas that are observed as white areas in the phase images.

[0060] The FESEM micrographs in FIG. 3 clearly show how a BAK salt (gray phase) microcoating is formed on the surface of the PET (darker phase) with a thickness of about 25 m, thereby confirming that the BAK compound adheres to the PET plastic. Furthermore, EDS analysis shows a 0.37% by weight nitrogen content in the BAK coating, that is consistent with the nitrogen atom present in the BAK compound and absent in the PET plastic. Therefore, EDS analysis in the PET matrix does not show this nitrogen content as was expected.

2.2. Opacity

[0061] FIG. 4 shows how there are not statistically significant differences in transparency (or opacity) of the PET disks before and after treatments with solvent or treatment with BAK, which is fundamental for the final use of the transparent protection equipment (face protection shields, protective shields, helmets, protective glasses, protective counter, transparent space divider, etc.) is essential. For quantitative results, opacity (O), which is an established measurement of transparency, was calculated using equation (1) and is shown in FIG. 4.

2.3. Antibacterial Activity

[0062] FIG. 5 shows the antibacterial results obtained from the untreated plastics (U plastic), the plastic treated with absolute ethanol/distilled water (70/30 v/v) referred to as S Plastic, and the plastic treated with 70% ethanol containing benzalkonium chloride (BAK Plastic) against multi-resistant bacteria MRSA and MRSE.

[0063] It can therefore be observed that plastics with the BAK biofunctional coating (BAK Plastic) showed powerful antibacterial activity against MRSA and MRSE with normalized antibacterial halos of 0.61±0.03 and 0.57±0.05, respectively.

2.4. Antiviral Activity

[0064] An RNA virus with an envelope was used as the biosafe viral model of the SARS-CoV-2 and other viruses with an envelope, such as the flu virus.

[0065] Phage phi6 is a double-stranded RNA virus segmented into three parts, a total of ˜ 13.5 kb long. Although this type of lytic bacteriophage belongs to group III in the Baltimore classification [Baltimore, D. Expression of animal virus genomes. Bacteriol. Rev. 1971, 35, 235-241], it was proposed in this case as a viral model of SARS-CoV-2, for biosafety reasons since it also has a lipid membrane around its nucleocapsid. Therefore, BAK plastic showed strong antiviral activity against this virus (100% viral inhibition, see FIGS. 3 and 4). The bacteria grew and no bald spots were observed after 1 minute of contact of the biosafe viral model of SARS-CoV-2 with the surface of the BAK plastic. Furthermore, the untreated plastic (U Plastic) and the plastic treated with the solvent without benzalkonium chloride (S Plastic) showed results similar to the control without antiviral activity (see FIGS. 6 and 7).

[0066] Phage titers of each type of sample were calculated and compared with the control (see FIG. 7 and Table 1 below).

TABLE-US-00001 TABLE 1 Phi6 phage titers a 1 minute of contact: control, untreated plastic (U Plastic), plastic treated with the solvent but without benzalkonium chloride (S Plastic), and plastic with biofunctional benzalkonium chloride coating (BAK Plastic) Phage phi6 at 1 min of Sample contact (PFU/mL) Control 4.36 × 10.sup.6 ± 2.92 × 10.sup.5 U Plastic 4.38 × 10.sup.6 ± 1.98 × 10.sup.5 S Plastic 4.23 × 10.sup.6 ± 1.36 × 10.sup.6 BAK Plastic 0.00 ± 0.00

[0067] FIG. 7 and Table 1 show that the titers obtained upon bringing the phages into contact with the untreated plastic (U Plastic) or the plastic treated with the solvent without benzalkonium chloride (U Plastic) are similar to the control. However, the plastic with the biofunctional benzalkonium chloride coating (BAK Plastic) showed strong phage inactivation.

[0068] The results obtained with SARS-Co-2 after 1 min of contact with the untreated plastic (U Plastic) or the plastic treated with the solvent without benzalkonium chloride (U Plastic) and the plastic with the biofunctional benzalkonium chloride coating (BAK Plastic) are shown in FIG. 8 and Table 2.

TABLE-US-00002 TABLE 2 SARS-CoV-2 virus titers at 1 minute of contact: untreated plastic (U Plastic), plastic treated with the solvent but without benzalkonium chloride (S Plastic), and plastic with biofunctional benzalkonium chloride coating (BAK Plastic) SARS-CoV-2 at 1 min Sample (PFU/mL) U Plastic 1.26 × 10.sup.5 ± 4.50 × 10.sup.4 S Plastic 8.54 × 10.sup.4 ± 2.53 × 10.sup.4 BAK Plastic 1.57 × 10.sup.4 ± 1.39 × 10.sup.4

[0069] These results clearly demonstrate that the plastic with BAK biofunctional coating (BAK Plastic) is very effective against SARS-CoV-2, inactivating more than 99% of the virus in 1 minute of contact. These results confirm the antiviral activity obtained by means of the Phi6 biosafe viral model used in this study (see FIGS. 6 and 7).

LITERATURE REFERENCES

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