Transparent Polymeric Formulations Effective against SARS-CoV-2 Infection.

The main route of the transmission of the SARS-CoV-2 virus is through airborne small aerosol particles containing viable virus as well as through droplets transmitted between people within close proximity. Transmission via contaminated surfaces has also been recognized as an important route for the spread of SARS-CoV-2 coronavirus. Among a variety of antimicrobial agents currently in use, polymers represent a class of biocides that have become increasingly important as an alternative to existing biocidal approaches. Two transparent polymeric compounds, containing silver and benzalkonium ions electrostatically bound to a polystyrene sulfonate backbone, were synthesized, through simple procedures, and evaluated for their antimicrobial properties against Gram-positive and Gram-negative bacteria and Candida albicans (ISO EN 1276) and for their antiviral activity toward 229E and SARS-CoV-2 coronaviruses (ISO UNI EN 14476:2019). The results showed that the two tested formulations are able to inhibit the growth of (1.5-5.5) × 1011 CFU of Gram-positive bacteria, Gram-negative bacteria, and of the fungal species Candida albicans. Both compounds were able to control the 229E and SARS-CoV-2 infection of a target cell in a time contact of 5 min, with a virucidal effect from 24 to 72 h postinfection, according to the European Medicines Agency (EMA) guidelines, where a product is considered virucidal upon achieving a reduction of 4 logarithms. This study observed a decrease of more than 5 logarithms, which implies that these formulations are likely ideal candidates for the realization of transparent surface coatings that are capable of maintaining remarkable antibacterial activity and SARS-CoV-2 antiviral properties over time.

Introduction

The global COVID-19 pandemic highlighted the need for innovative methods and technologies to mitigate the spread of viruses. The main route of the transmission of the SARS-CoV-2 virus occurs through small, airborne aerosol particles containing viable virus as well as through larger droplets that are transmitted between people within close proximity.[1] Transmission via contaminated surfaces has also been recognized as an important route for the spread of SARS-CoV-2 coronavirus, and several approaches to produce antimicrobial surfaces have been recently explored.[2,3]

Notable results have been obtained with nanostructured surfaces,[4,5] surfaces coated with copper oxide,[6,7] and surface coated with cuprous oxide particles bound with polyurethane.[8]

Among a variety of available antimicrobial agents, polymers represent a class of biocides that have become increasingly important as an alternative to existing biocides. Conventional disinfectants are based on low-molecular-weight liquids or gases, so their use and duration are restricted by their volatility. Antimicrobial polymers can be tethered to surfaces enabling the killing of microbes without releasing biocides. Many of the previously reported antimicrobial polymers have focused on antibacterial capabilities; however, there has been much less focus on antiviral properties. Antimicrobial polymers have been known since 1965, when Cornell and Donaruma described polymers and copolymers that kill bacteria.[9] Since then, a number of reports appeared in the literature describing bactericidal coatings incorporating bound[8] or unbound biocidal agents, such as metal-oxide particles,[10] chlorine dioxide,[11] iodine compounds,[12,13] silver nanoparticles,[14] benzophenone,[15] or quaternary ammonium (QA) compounds[16,17] mixed with a polymeric binder. Among these coatings, QA compounds are probably the most widely used antibacterial agents for medical and public health applications because they have been shown to be effective against both Gram-negative and Gram-positive bacteria.[18]

According to the literature,[19] there are three general types of antimicrobial polymer: (1) polymeric biocides, where the repeating unit is a biocide; (2) biocidal polymers, where the active unit is randomly embedded within a macromolecule, and (3) biocide-releasing polymers where the polymeric backbone acts as a carrier support for the biocides that can be transferred to the cell. In addition to these classic polymers, photoactive polymers and oligomers have recently demonstrated highly effective light-induced inactivation of SARS-CoV-2, which may cause a 5-log reduction in pfu/mL within 10 min of irradiation.[20]

Our work has focused on simple polymeric compounds where an active principle can be electrostatically bound to the backbone of a water-soluble anionic polymer, resulting in the formation of water-insoluble polymeric materials, which can be then solubilized in alcoholic solvents. In this first study, we have considered silver-containing species and benzalkonium ions electrostatically bound to polystyrene sulfonate. The alcoholic solutions of these polymeric materials are ideal for realizing antimicrobial coatings on everyday items handled by people such as doorknobs, keyboards, handles, as well as on any type of filtration system where the polymeric backbone adheres to the solid substrate because of significant van der Waals interactions. The resulting films are transparent and are expected to exhibit a long-lasting antimicrobial effect.

The observation that the coated materials are highly effective against Gram-positive and Gram-negative bacteria and Candida albicans prompted us to investigate the antiviral activity of the solid polymeric film because viruses can also survive for a period of hours to several days once established on inanimate surfaces. We report here strong evidence of the activity of polymeric films against 229E and SARS-CoV-2 coronaviruses.

Materials and Methods

Materials

All chemical products were purchased from Sigma-Aldrich. Elemental analysis C,H,N was performed with a LECO CHN analyzer. Silver was determined through plasma atomic absorption spectrometry with a Perkin Elmer Optima 3100 XL. Fourier transform infrared (FT-IR) spectra were recorded on a Bruker Vertex 70 FT-IR in diffuse reflectance mode dispersing the chemical powder in KBr (potassium bromide). Dimensional analysis and zeta potential were obtained with a Z-sizer Malvern instrument.

Preparation

The preparation of the silver complex, complex salts, and micellar derivatives was first described in the patent application PCT/EP2020/060686.

Na3[Ag(MPA)2]( MPA = 2-Mercapto-4-methyl-5-thiazolacetate). To a 1.135 g amount of the protonated MPA (2-mercapto-4-methyl-5-thiazolacetic acid, Aldrich), 12.36 g of 1 M NaOH solution in water was added. Distilled water (186.5 g) was then added, and the solution was kept stirring for 5 min. An aqueous solution (100 g) containing 0.51 g of di AgNO3 was finally added, and the yellow solution was kept under stirring for an additional 30 min. The Na3[Ag(MPA)2] complex salt was precipitated by the addition of an excess of acetone, filtered, and air-dried; Ag+ % = 0.108%. Elemental analysis: calculated for Na3AgC12N2S4O4H10; C 26.14; N, 5.08; H, 1.83; Ag, 19.57. Found: C, 25.87; N 5.0; H 1. 88; Ag 19.46.

[Ag(MPA)2](Bz)3 (Bz = Benzalkonium) Micellar Derivatives. Benzalkonium (Bz) CAS. n 63,449–41-2 is a mixture of alkylbenzyldimethylammonium chlorides, in which the alkyl group has various even-numbered alkyl chains from 8 to 18. The average molecular weight of the chloride salt is 370 g. Uncharged [Ag(MPA)2](Bz)3 salt can be easily precipitated by the addition of a slight excess of benzalkonium chloride to an aqueous solution of the silver complex Na3[Ag(MPA)2]. The addition of an excess of benzalkonium chloride results, instead, in the dissolution of [Ag(MPA)2](Bz)3 because of the formation of positively charged micellar derivatives with a size distribution in the range 10–500 nm (see the next section).

Polymeric salts PSS-Bz-Ag and PSS-Bz. Polymeric salts of the positively charged micellar derivatives of [Ag(MPA)2](Bz)3 or Bz ions were obtained in distilled water by the addition of sodium polystyrene sulfonate (PSS). The precipitated neutral polymers containing PSS, Ag(MPA)2, and Bz moieties (PSS-Bz-Ag) or PSS and Bz ions (PSS-Bz) were separated from the aqueous solutions by filtration, washed with water, and dried at 50 °C. The polymeric solid was then dissolved in isopropanol or ethanol to obtain a solution of PSS-Bz-Ag containing Bz 1.25%, PSS 0.62%, and Ag 0.0025% and a solution of PSS-Bz containing Bz 1.25% and PSS 0.62%.

Bacterial Strains

The antimicrobial activity of the liquid products PSS-Bz-Ag (composition: Bz 1.25%, Ag+ 0.0025%, PSS 0.62%, ethanol) and PSS-Bz (composition: Bz 1.25%, PSS 0.62%, ethanol) was tested against the following strains of microorganisms (purchased from Diagnostic International Distribution S.p.A.): Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, Enterococcus hirae ATCC 10541, Candida albicans ATCC 10231, in accordance with ISO EN 1276. Microbial suspensions were prepared according to EN1276 from lyophilized pellets resuspended in the LB (Luria Broth) medium. The concentration was determined by spectrophotometry, horizontal method, and biochemical test. Microbial suspensions of the different strains were prepared with concentrations expressed as colony-forming units (CFU) comprised between 1.5 × 1012 and 5.5 × 1012 for each species. Samples (100 μL) of the two products were deposited in the center of Petri dishes and dried for 2 h in an oven at 37 °C. The microbial pool (100 μL) was then added and left in contact with PSS-Bz-Ag or PSS-Bz for 5 min. TSA (tryptone soya agar) was added after cooling at 40 °C and incubated at 37 °C for 24 h. Microbial titration was determined using the horizontal method. The experiments were performed in triplicate.

Cells and Viruses

MRC5 human fibroblast cells (ATCC CCL171) and African green monkey epithelial kidney cells, VeroE6 (ATCC CRL1586), were grown in monolayers in Eagle’s minimal essential medium (MEM) with nonessential amino acids (Lonza Biosciences; Italy) supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco-Life Technologies; Italy), penicillin (100 units/mL; Sigma-Aldrich), streptomycin (100 μg/mL; Sigma-Aldrich; Italy), and l-glutamine (2 mM; Sigma-Aldrich; Italy).

Human coronavirus 229E (ATCC VR-740) and the clinical isolate of SARS-CoV-2, kindly provided by Prof. A. Caruso and Prof. F. Caccuri, University of Brescia, Italy, were propagated and titrated by qPCR assay on MRC5 or VeroE6 cells, respectively.[21,22] All procedures were carried out in a biosafety level-3 (BSL-3) laboratory.

Cytotoxicity Control

Cytotoxicity control was performed on MRC5 and VeroE6 cells. The culture medium was mixed with PSS-Bz-Ag, PSS-Bz, or PBS (phosphate-buffered saline, 1X control solution) (Gibco, USA). The resulting solutions were added to cell cultures and incubated for 5, 10, 15 min, 24, 48, and 72 h. Cell viability was examined by the MTT (3-(4,5-dimethilthiazol-2yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay (Roche Diagnostics Corporation, Indianapolis, IN) and trypan blue dye (Sigma-Aldrich; Italy) exclusion. 2% DMSO (dimethyl sulfoxide; Sigma-Aldrich; USA) was used as the positive control of cell cytotoxicity. The experiments were performed in triplicate.

Antiviral Assays

The antiviral effect on the 229E and SARS-CoV-2 infection was evaluated by quantitative assays (plaque assay), in accordance with ISO UNI EN 14476:2019.

The 229E and human coronavirus SARS-Cov-2 suspensions were prepared by infecting the monolayers of MRC5 and VeroE6 cells, respectively. The infections were performed at 1.0 multiplicity of infections (MOIs).[20]

On the day of testing, sterile Petri dishes (35 mm diameter) were prepared, as previously reported (Section ). The viral suspension was added to the PSS-Bz-Ag, PSS-Bz, or PBS (control)-treated Petri dishes and incubated for 5 min at room temperature. The viral suspension was then harvested and incubated with susceptible cells for 1 h at 37 °C to let the viral particle infect the cells. The virus inoculum was then removed, the cells were washed, and 1 mL of EMEM 2% FCS was added. The ability of the viral particles to infect susceptible cells was assessed on cell culture supernatants 24, 48, and 72 h postinfection (p.i.). Viral titration was performed by plaque assay and quantitative reverse transcription (qRT)-PCR.

Plaque Assay

Cell culture supernatants were harvested and placed in an ice bath immediately. The samples were inoculated in cell cultures immediately, within 10 s. Five days after infection, cells were methanol-fixed, and plaques were stained with crystal violet (0.1%) and counted. The experiments were performed in triplicate.

Quantitative Reverse Transcription-PCR

RNA was extracted from clarified cell culture supernatants (16,000 g × 10 min) using PureLink Viral RNA/DNA Mini Kit (Invitrogen) according to the manufacturer’s instructions. RNA was eluted in 15 μL of RNase-free water and stored at −80 °C until use. Briefly, RNA was reverse-transcripted with SuperScript IV VILO (Invitrogen). 229E quantification was performed by real-time PCR with an HCoV-229E-specific qPCR gene assay (Vi06439671_s1, Catalog number: 4,331,182, ThermoFisher).[19] SARS-CoV-2 quantification and amplification of the S gene were performed using the PowerUp SYBR Green Master Mix (Applied Biosystems) as follows: primers: RBD-qF1: 5′-CAATGGTTTAACAGGCACAGG-3′ and RBD-qR1: 5′-CTCAAGTGTCTGTGGATCACG-3′.[20] A standard curve was generated by the determination of copy numbers derived from serial dilutions (102–108 copies) of the corresponding gene block (IDT Technologies). Each quantification was run in triplicates.

After-Effect Control

After-effect controls were conducted by mixing cell suspension with PSS-Bz-Ag, PSS-Bz, or PBS (10 s, see Section ). The cells were then harvested and infected with viral inoculum for 1 h at 37 °C. The viral suspension was then incubated with susceptible cells for 1 h at 37 °C to allow for the viral particles to infect the cells. The virus inoculum was then removed, the cells were washed, and 1 mL of EMEM 2% FCS was added. The ability of the viral particles to infect susceptible cells was assessed on cell culture supernatants 24, 48, and 72 h postinfection (p.i.) by plaque assay. The experiments were performed in triplicate.

Interference Control

On the day of testing, sterile Petri dishes (35 mm diameter) were prepared, as previously reported (Section ). Cell suspensions were added to the treated Petri dishes (PSS-Bz-Ag, PSS-Bz, or PBS (control)) for 1 h at room temperature, corresponding to the time period of the viral inoculum contact. The cells were then harvested and infected with a viral suspension (1.0 m.o.i.) for 1 h. The ability of the viral particles to infect susceptible cells was assessed on cell culture supernatants 24, 48, and 72 h postinfection (p.i.) by plaque assay. The experiments were performed in triplicate.

Statistical Analysis

Statistical comparisons of the data of the control and treatment groups were performed using Student’s t-test as the data displayed a normal distribution based on the Kolmogorov–Smirnov test. Differences were considered significant at p < 0.05. Statistical analyses were performed using GraphPad Prism version 9.

Results and Discussion

PSS-Bz-Ag and PSS-Bz Characterization

Elemental analysis and spectroscopic data for the silver complex were consistent with the presence of two MPA anionic ligands coordinated to Ag+ ions. The FT-IR spectrum of MPA showed the strong CO-stretching band of the carboxylic function at 1704 ± 2 cm–1 and an S-H stretching band at 2555 ± 2 cm–1; this band disappeared in the anionic silver complex, consistent with the coordination of sulfur to Ag+. Intense bands due to asymmetric and symmetric stretching modes of carboxylate appeared at 1580 ± 2 and 1386 ± 2 cm–1, respectively.

The addition of benzalkonium chloride to the anionic silver complex Ag(MPA)23–, in water, resulted in the neutralization of the negative charge with the formation of a precipitate consistent with the stoichiometry (Bz)3[Ag(MPA)2]. The salt could be solubilized in water by the addition of an excess of benzalkonium chloride to form micelles with diameters of the order of 2–3 nm and a zeta potential of ca. + 9 mV. Subsequent addition of PSS to the micellar product allowed to obtain a polymeric material, PSS-Bz-Ag, insoluble in water, but exceedingly soluble in alcoholic solvents. Analogous polymers, PSS-Bz, were obtained by the addition of an excess of benzalkonium chloride to aqueous solutions of PSS. The two polymers did not show any appreciable water solubility, as shown by the analysis of the electronic absorption spectra of filtered suspensions and by the lack of the antimicrobial activity of the LB or Eagle MEM culture media left in contact for 20 h with the solid polymers (see solubility of the polymeric PSS-Bz-Ag and PSS-Bz components and Figure S1 and S2 in the Supporting Information).

The IR spectra of solid PSS-Bz-Ag and PSS-Bz (Figure S3) were similar, with vibrational bands matching the superposition of the intense bands of benzalkonium and PSS.

Solutions of PSS-Bz-Ag in alcohol were composed of micellar aggregates with diameters of the order of 10 nm and zeta potential of −3.9 mV (Figure S4a). Minority aggregates with higher diameters could be observed in the size distribution by intensity spectra (Figure S4b).

Films of PSS-Bz-Ag and PSS-Bz polymers can be produced on any type of surface by spray coating or wiping the alcoholic solutions that evaporate quickly, leaving a very transparent layer, as shown in Figure .

Figure 1

Glass slides untreated and treated with PSS-Bz-Ag and PSS-Bz.

Antimicrobial Activity of PSS-Bz-Ag and PSS-Bz Compounds

To assess the antimicrobial activity of PSS-Bz-Ag and PSS-Bz, 100 μL of the two products was deposited on a plastic substrate and allowed to dry. The microbial pool (100 μL) was then added and left to be in contact with PSS-Bz-Ag or PSS-Bz for 5 min. We used different dilutions of the microbial suspension, with a range of 1.5 × 1012 and 5.5 × 1012 CFU for each species. As shown in Figure , while in the control dish, there is a continuum of colonies (Figure a), we observed no colonies in the Petri dishes of PSS-Bz-Ag- or PSS-Bz-treated microbial suspensions (Figure b,c), indicating a complete antimicrobial activity of both PSS-Bz-Ag and PSS-Bz that is comparable to isopropanol 80%, used as the positive control (Figure d). The analytical result obtained indicates that the two formulations are able to inhibit the growth of (1.5–5.5) × 1011 CFU of Gram-positive bacteria, Gram-negative bacteria, and the fungal species Candida albicans (Figure e). At higher concentrations, we observed the appearance of some colonies in isopropanol 80% (1 × 107 CFU) and, to a lesser extent, in PSS-Bz-Ag- (1 × 106 CFU) and PSS-Bz (1.2 × 106 CFU)-treated samples.

Figure 2

Antimicrobial activity of PSS-Bz-Ag and PSS-Bz. Representative cultures of microbial suspensions: (a) PBS (control), (b) PSS-Bz-Ag, c) PSS-Bz, or (d) isopropanol 80% treated. (e) Analytical results on different microbial suspensions, containing Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterococcus hirae, and Candida albicans (concentrations 1: 1.5 × 1011; 2: 2.0 × 1011; 3: 5.0 × 1011; 4: 1.5 × 1012; 5: 2.0 × 1012; and 6: 5.5 × 1012 for each species). Data are reported as mean ± standard deviation of three replicates. *p value <0.001; Student’s t-test.

Cytotoxicity and Interference Tests for PSS-Bz-Ag and PSS-Bz Compounds

First, we assessed the cytotoxic effect of PSS-Bz-Ag or PSS-Bz toward human cells. We mixed the culture medium with PSS-Bz-Ag or PSS-Bz, and we used MRC5 and VeroE6 cell lines to perform the cytotoxic assay (MTT). We sprayed a plastic surface with PSS-Bz-Ag, PSS-Bz, or PBS and added the resulting solutions to MRC5 or VeroE6 cells for 5, 10, 15 min and 1, 24, 48, and 72 h. We observed that both PSS-Bz-Ag and PSS-Bz were not cytotoxic for human cells. In fact, the MTT assay (Figure a–d) and the viable cell count (Figure e–h) did not show any significant difference in comparison with PBS-treated cells (p: NS; Student’s t-test).

Figure 3

Cytotoxicity effect of the PSS-Bz-Ag compound on (a, b) MRC5 and (c, d) VeroE6 cell lines and PSS-Bz compounds on (e, f) MRC5 and (g, h) VeroE6 cell lines. (i) SARS-CoV-2 replication in VeroE6 cells after treatment with PSS-Bz-Ag and PSS-Bz compounds, 48 h postinfection. Data are reported as mean +/– standard deviation of three replicates.

The interference control was performed by adding to cell cultures with PSS-Bz-Ag, PSS-Bz, or PBS for 1 h. PSS-Bz-Ag, PSS-Bz, or PBS solutions were removed, and the cells were infected with viral inoculum (1.0 m.o.i.) for 1 h. Viral titration showed no interference effect; in fact, viral plaques in PBS-treated samples did not differ from PSS-Bz-Ag- or PSS-Bz-treated samples (p: NS; Student’s t-test) (Table ).

Table 1

Interference Testa

	logarithmic reduction (LR) (pfu/mL)
time postinfection (h)	PSS-Bz-Ag	PSS-Bz
229E
24	0.3 ± 0.1	0.3 ± 0.1
48	0.3 ± 0.1	0.2 ± 0.1
72	0.2 ± 0.2	0.1 ± 0.2
SARS-CoV-2
24	0.3 ± 0.1	0.3 ± 0.1
48	0.2 ± 0.2	0.2 ± 0.1
72	0.2 ± 0.1	0.1 ± 0.2

LR of 229E and SARS-CoV-2 viral plaques after PSS-Bz-Ag, PSS-Bz, or Ag treatment in comparison with PBS-treated control. Data are reported as mean +/– standard deviation of three replicates.

Antiviral Test for PSS-Bz-Ag and PSS-Bz Compounds

We tested the anti-229E and anti-SARS-CoV-2 effect of both PSS-Bz-Ag and PSS-Bz. The viral inoculum was exposed to a treated surface with PSS-Bz-Ag, PSS-Bz, or PBS. The time of contact of the viral inoculum was 5 min at room temperature, and then, the viral inoculum was recovered and used to infect MRC5 and VeroE6 cells and the target cells for 229E and SARS-CoV-2 in vitro assays. The viral inoculum was recovered after 1 h, and the cells were analyzed for viral titer 24, 48, and 72 h postinfection. These time points were selected as they allowed the detection of viral progeny formation, suggestive of viral infectivity. The viral load was determined by plaque assay. We observed a decreased viral plaque count after the treatment with both compounds in both cell lines, as reported in Table . At all time points, we observed a > 5 logarithmic reduction (LR) of plaque formation in 229E-infected samples treated with PSS-Bz-Ag or PSS-Bz (p < 0.001; Student’s t-test). SARS-CoV-2-infected samples showed a > 5 LR of plaque formation in the PSS-Bz-treated samples (p < 0.001; Student’s t-test). The PSS-Bz-Ag treatment was less effective at 24 h postinfection (p = 0.02; Student’s t-test) and reached a > 5 LR of plaque formation after 48–72 h postinfection (p < 0.001; Student’s t-test).

Table 2

LR of 229E and SARS-CoV-2 Viral Plaques after PSS-Bz-Ag, PSS-Bz, or Ag Treatment in Comparison with the PBS-Treated Controla

	LR (pfu/mL)
time post-infection (h)	PSS-Bz-Ag	PSS-Bz	Ag 0.0025%	Ag 0.125%
229E
24	5.9 ± 0.3	6.1 ± 0.5	0.2 ± 0.3	0.4 ± 0.3
48	5.9 ± 0.8	5.9 ± 0.6	0.3 ± 0.4	0.4 ± 0.4
72 h	5.2 ± 0.6	5.9 ± 0.4	0.2 ± 0.3	0.3 ± 0.3
SARS-CoV-2
24	3.8 ± 0.6	5.3 ± 0.5	0.9 ± 0.5	0.9 ± 0.4
48	5.0 ± 0.3	5.5 ± 0.5	0.8 ± 0.6	0.8 ± 0.5
72	>7 ± 0.5	5.4 ± 0.4	0.8 ± 0.5	0.8 ± 0.5

Data are reported as mean ± standard deviation of three replicates.

To obtain a further confirmation of the results obtained using the plaque formation test, we evaluated viral RNA by RT-qPCR in the culture medium collected from infected MRC5 and VeroE6 cells. We observed a decreased viral load after the treatment with both compounds in both cell lines (Table ).

Table 3

LR of 229E and SARS-CoV-2 Viral RNA after PSS-Bz-Ag, PSS-Bz, or Ag Treatmenta

	LR genome copies/mL
time postinfection (h)	PSS-Bz-Ag	PSS-Bz	Ag 0.0025%	Ag 0.108%
229E
24	6.1 ± 0.6	6.5 ± 0.4	0.5 ± 0.3	0.6 ± 0.4
48	6.0 ± 0.3	6.4 ± 0.2	0.5 ± 0.2	0.6 ± 0.3
72	5.9 ± 0.4	6.3 ± 0.5	0.5 ± 0.2	0.6 ± 0.2
SARS-CoV-2
24	3.7 ± 0.2	5.2 ± 0.4	0.8 ± 0.2	0.9 ± 0.3
48	4.9 ± 0.5	5.3 ± 0.2	0.7 ± 0.2	0.8 ± 0.2
72	6.8 ± 0.3	5.2 ± 0.1	0.6 ± 0.3	0.8 ± 0.3

Data are reported as mean+/– standard deviation of three replicates.

The results showed that PSS-Bz-Ag led to a 6.1 ± 0.6 LR for 229E RNA copies and a 3.7 ± 0.2 LR for SAS-CoV-2 RNA copies at 24 h postinfection (Table ) (p < 0.001; p = 0.012, respectively; Student’s t-test). We observed a similar reduction of 229E RNA copies at different time points, while SARS-CoV-2 RNA copies were significantly reduced over time (24 vs 48 h p < 0.021; 24 vs 72 h p < 0.001; 48 vs 72 h p = 0.019; Student’s t-test). PSS-Bz led to a 6.5 ± 0.4 LR for 229E RNA copies and a 5.2 ± 0.4 LR for SARS-CoV-2 RNA copies at 24 h postinfection (Table ). We observed a similar reduction of 229E and SARS-CoV-2 RNA copies over time (Table ). These results confirm that both PSS-Bz and PSS-Bz-Ag are able to control 229E and SARS-CoV-2 infection of a target cell with a virucidal effect 24 h postinfection, according to the EMA guidelines,[23] where a product is considered virucidal as soon as it has achieved a reduction of ≥4 logarithm.

To evaluate the possible after-effect of PSS-Bz and PSS-Bz-Ag, cell suspensions were added to the treated Petri dishes (PSS-Bz-Ag, PSS-Bz, or PBS (control)) for 1 h at room temperature, corresponding to the time period of the viral inoculum contact. The cells were then harvested and infected with a viral suspension (1.0 m.o.i.) for 1 h. The ability of the viral particles to infect susceptible cells was comparable to that of control samples (PBS-treated) at all time points (Table ), demonstrating that both PSS-Bz and PSS-Bz-Ag are effective on viral particles and not on human cells.

Table 4

After-Effect Testa

	LR (pfu/mL)
time postinfection (h)	PSS-Bz-Ag	PSS-Bz
229E
24	0.2 ± 0.1	0.1 ± 0.1
48	0.1 ± 0.2	0.1 ± 0.1
72	0.1 ± 0.2	0.1 ± 0.1
SARS-CoV-2
24	0.2 ± 0.1	0.1 ± 0.1
48	0.1 ± 0.2	0.1 ± 0.1
72	0.1 ± 0.1	0.1 ± 0.1

Logarithmic reduction of 229E and SARS-CoV-2 viral plaques after PSS-Bz-Ag, PSS-Bz, or Ag treatment in comparison with the PBS-treated control. Data are reported as mean ± standard deviation of three replicates.

Because both the main difference between PSS-Bz-Ag and PSS-Bz compounds is the presence of Ag in PSS-Bz-Ag, we evaluated the effect of the Ag complex alone at two concentrations, one corresponding to the concentration in PSS-Bz-Ag (0.0025% w/w and a 50 times higher concentration (0.125% w/w) on 229E and SARS-CoV-2 infection. The Ag treatment had a slight effect on viral plaque formation, with both Ag concentrations (Table ). Interestingly, the effect is not concentration- and time-dependent (Table ). RNA copy analysis confirmed the plaque assay results (Table ), showing a slight LR of RNA copies with both Ag concentrations. These results support the hypothesis that the main virucidal component is benzalkonium chloride. Literature data support this efficacy based on the cationic “headgroup” of benzalkonium chloride that is progressively adsorbed to the negatively charged phosphate heads of phospholipids in the lipid bilayer. This might be effective mainly on enveloped viruses as SARS-CoV-2, together with the alkyl chain “tail” component of benzalkonium chloride, that perturbs the membrane bilayer by permeating the barrier and disrupting its physical and biochemical properties. Protein function is subsequently disturbed, and the combination of these effects results in the solubilization of the bilayer constituents into benzalkonium chloride/phospholipid micelles.

Multiple studies have reported on the virucidal effect of Benzalkonium chloride against coronaviruses. Rabenau et al. found that, as a surface disinfectant in a concentration of 0.5%, benzalkonium chloride demonstrated a reduction factor of >4 against SARS-CoV.[24] Meanwhile, by evaluating the virucidal activity of different oral rinses against three strains of SARS-CoV-2, Meister et al. reported log reductions of >3.1, >2.8, and > 2.6, respectively, for a rinse containing 0.035% benzalkonium chloride.[25]

Interestingly, PSS-Bz-Ag and PSS-Bz showed a different efficacy toward the two viral strains. In particular, both PSS-Bz-Ag and PSS-Bz affected 229E infectivity with a similar efficacy during culture time points (∼6 LR). PSS-Bz strongly affected SARS-CoV-2 infectivity at all culture time points (>6 LR). On the contrary, the PSS-Bz-Ag effect is time-dependent, increasing from about 4 to 7 LR at 24 and 72 h culture, respectively. This different behavior is unclear at present, and it might be due to the differences in the PSS-Bz-Ag and PSS-Bz structures, which are illustrated in Figure a,b.

Figure 4

(a) PSS-Bz-Ag and (b) PSS-Bz schematic structures of the polymeric assemblies.

In PSS-Bz-Ag, the synthetic design obliges positively charged Bz micellar assemblies, trapping the neutral [Ag(MPA)2](Bz)3 complex salt, to interact electrostatically with the sulfonic groups of PSS (Figure )a. The electrostatic interaction is strong because the polymer does not dissolve in the polar water solvent, but the alkyl chain of Bz is mainly engaged in the solubilization of the neutral complex salt. In PSS-Bz, on the contrary, the direct electrostatic interaction of the quaternary nitrogen with negative sulfonates leaves the alkyl chain completely free from interacting with the microbial membrane, as shown in Figure b. Surface coatings with PSS-Bz should cause a higher damage to the virus most probably for this reason, in fact, it is generally accepted that the mechanism of the microbicidal action of QA compounds is due to the dissociation of cellular membrane lipid bilayers, which compromises cellular permeability controls and induces the leakage of cellular contents. We might hypothesize that PSS-Bz is able to interfere with the SARS-CoV-2-cell contact, reducing infectivity at all time points, while PSS-Bz-Ag might interfere with SARS-CoV-2 replication, with a time-dependent effect. The strong efficacy of both compounds on 229E infection supports the different resistances of these two coronaviruses.[26] The results obtained with interference and after-effect tests confirm that both compounds are effective during the 5 min of contact, and they are not removed within the viral inoculum. The water insolubility of these products should allow and help maintain their effectiveness over time.

Conclusions

We have examined the antibacterial and antiviral properties of polymeric compounds that can be easily synthesized by coupling positively charged antimicrobial agents (either as a cationic molecule or metal complex) with a negatively charged polymer such as sulfonated polystyrene. Our general approach has been to produce a water-insoluble material, which could be solubilized in a volatile organic solvent for spray-coating surfaces. Specifically, we have analyzed the properties of the transparent polymeric formulations consisting of polystyrene sulfonate, silver, and benzalkonium or simply by polystyrene sulfonate and benzalkonium. These studies support a very high antibacterial and antiviral effect toward 229E and SARS-CoV-2, as evidenced by a > 5 LR in the infectious agent after 5 min of contact with the treated surface. The reported examples can be useful for the production of new polymeric materials containing different cationic antimicrobial species.