Anti-influenza Activity of Povidone-Iodine-Integrated Materials

Abstract

Personal protective equipment (PPE), including medical masks, should be worn for preventing the transmission of respiratory pathogens via infective droplets and aerosols. In medical masks, the key layer is the filter layer, and the melt-blown nonwoven fabric (NWF) is the most used fabric. However, the NWF filter layer cannot kill or inactivate the pathogens spread via droplets and aerosols. Povidone-iodine (PVP-I) has been used as an antiseptic solution given its potent broad-spectrum activity against pathogens. To develop PPE (e.g., medical masks) with anti-pathogenic activity, we integrated PVP-I into nylon-66 NWF. We then evaluated its antiviral activity against influenza A viruses by examining the viability of Madin–Darby canine kidney (MDCK) cells after inoculation with the virus strains exposed to the PVP-I-integrated nylon-66 NWF. The PVP-I nylon-66 NWF protected the MDCK cells from viral infection in a PVP-I concentration-dependent manner. Subsequently, we found to integrate PVP-I into nylon-66 and polyurethane materials among various materials. These PVP-I materials were also effective against influenza virus infection, and treatment with PVP-I nylon-66 NWF showed the highest cell survival among all the tested materials. PVP-I showed anti-influenza A virus activity when used in conjunction with PPE materials. Moreover, nylon-66 NWF integrated with PVP-I was found to be the best material to ensure anti-influenza activity. Therefore, PVP-I-integrated masks could have the potential to inhibit respiratory virus infection. Our results provide new information for developing multi-functional PPEs with anti-viral activity by integrating them with PVP-I to prevent the potential transmission of respiratory viruses.

INTRODUCTION

Influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are transmitted from human-to-human via infective droplets and aerosols that are generated when a virus-infected person speaks, coughs, or sneezes.^1,2) These infectious droplets and aerosols can easily access the mucosal surfaces of the respiratory tract of persons who are near the source via the nose and/or mouth. To prevent the transmission of these viruses, it is important to wear personal protective equipment (PPE), including face masks, respirators, gloves, hats, earplugs, vests, and body suits, in healthcare settings. In fact, the Center for Disease Control and Prevention (https://www.cdc.gov/flu/professionals/infectioncontrol/maskguidance.htm and https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/masks.html) and the WHO (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-public/when-and-how-to-use-masks) recommend wearing face masks or respirators for preventing the spread and transmission of influenza virus and SARS-CoV-2 infection in the public, private, and healthcare settings. Ueki et al. recently reported that cotton masks with four layers of gauze, surgical masks, or N95 masks protected the wearers from the transmission of infective SARS-CoV-2 droplets and aerosols in airborne simulation experiments; however, surgical masks and N95 masks could not completely block the viral transmission via droplets and aerosols even when were fitted to face.³⁾ Thus, the filtration efficiency of common medical masks (e.g., surgical masks and N95 level respirators) may be insufficient for blocking the airborne transmission of virus droplets and aerosols.

The filter layer is key to the function of masks. It filters out droplets and aerosols containing pathogenic microorganisms (e.g., bacteria and virus), pollen, and particle matter <2.5 µm in diameter. Melt-blown nonwoven fabric (NWF) is the most used fabric to produce the filter layer of medical masks.⁴⁾ This material is fabricated from synthetic or natural polymers composites such as polypropylene (PP), polyethylene (PE), polyamide (nylon), glass papers, or woolen felt, using melt-blowing technique.⁴⁾ Medical masks composed of a NWF in the filter layer have shown >95% filtration efficiency for aerosol particles when well fitted.⁴⁾ However, this filter material cannot kill or inactivate pathogens spread via droplets and aerosols, and the mask with captured pathogens in its filter layer would inhibit the blocking of the airborne transmission of pathogens and become fomites causing secondary transmission or cross-infection. Hence, to resolve this problem, multi-functional medical masks with anti-respiratory pathogenic components integrated in the NWF filter layer are required.

Povidone-iodine (polyvinylpyrrolidone iodine complex, PVP-I) solution was developed in the nineteenth century and has been widely used as an antiseptic solution for more than 60 years given its potent broad-spectrum activity against bacteria, virus, fungus, and protozoa.^5,6) Further, 10% PVP-I solution, equivalent to 1% available iodine, is on the WHO Model List of Essential Medicines, which identifies important medicines necessary for a functional healthcare system. PVP-I solution effectively inactivates various enveloped and nonenveloped viruses, such as polio-, adeno-, rota-, mumps, herpes, and human immunodeficiency viruses,⁷⁾ with a short contact time relative to other available antiseptic agents.⁸⁾ Anti-influenza^9,10) and anti-SARS-CoV-2^11–13) activities of PVP-I have also been reported. PVP-I products, such as topical solution, scrub, gargle solution, or throat spray, in the range of 0.23–2%⁹⁾ and 0.45–10%^11,13–15) strongly reduced infectious titers of influenza virus and SARS-CoV-2, respectively. Therefore, PVP-I solution could be used as an anti-viral component integrated into the NWF filter layer for manufacturing medical masks with anti-viral activity.

In this study, we integrated PVP-I into nylon-66 NWF and evaluated its antiviral activity against influenza A viruses. We examined the viability of Madin–Darby canine kidney (MDCK) cells using naphthol blue-black (NB) staining and thiazolyl blue tetrazolium bromide (MTT) assay after inoculation with the virus strains exposed to the PVP-I-integrated nylon-66 NWF. We also integrated PVP-I into various materials and evaluated their antiviral activities against influenza A.

MATERIALS AND METHODS

Preparation of 5 and 20% PVP-I Ethanol Solutions

First, 25 g of PVP-I was gradually added to 25 mL of ethanol with stirring at 25 °C. After complete dissolution, 475 mL of purified water was added to prepare a 5% PVP-I ethanol solution; subsequently, 20 g of PVP-I was gradually added to 20 mL of ethanol with stirring at 25 °C. After complete dissolution, 80 mL of purified water was added to prepare a 20% PVP-I ethanol solution.

Preparation of PVP-I·Nylon-66 NWF Complexes

Nylon-66 NWF (#PO303WTO, UNITIKA, Tokyo, Japan) sandwiched between PP mesh sheets was gently stirred in 600 mL of 1% PVP-I ethanol solution at room temperature; after 6 min, the NWF was removed from the solution and dipped in 3 L of water for 10 s. This procedure was repeated with 3 L of purified water. After blotting the water using a paper towel, the NWF was air-dried and weighted (0.24 g). The iodine content of the NWF, determined by back titration with sodium thiosulfate solution, was 10–30 µg/cm². PVP-I·nylon-66 NWF complexes (PVP-I nylon-66 NWF) with PVP-I content of 50–100 µg/cm² were prepared in the same manner (Table 1).

Table 1. Conditions for Preparing PVP-I-Integrated Nylon-66 NWF

Material (abbreviation and manufacturer)	Concentration of PVP-I in an ethanol solution (%)	Reaction time (min)	Iodine weight per area of PVP-I-integrated NWF (µg/cm2)
Nylon-66 nonwoven fabrics (NWF) (#PO303WTO, UNITIKA, Tokyo, Japan)	1	6	10–30
	5	10	50–100

Integration with PVP-I into Various Materials

Polyester NWF (#DM105A, Kurashiki Textile Manufacturing, Osaka, Japan), rayon NWF (#EWK-3043, Japan Vilene Company, Tokyo, Japan), nylon-66 NWF (UNITIKA), polyolefin NWF (#DM35KW, SHINWA, Ehime, Japan), PP NWF (#6620-1A, SHINWA), PE woven fabric (WF) (#KIN-KG60777, WAVOU, Tokyo, Japan), nylon-66 WF (#SMD-FR30, WAVOU), polyester WF (#T3010, UTAX, Hyogo, Japan), cotton WF (#11WSR-456, WAVOU), rayon (#9060, Inami, Tokyo, Japan), nylon-66 film (NF) (#NO-25, UNITIKA), polyurethane film (PF) (#ET85, Okura Industrial, Kagawa, Japan), PE terephthalate film (#vurex film A31, TEIJIN LIMITED, Osaka, Japan), or PE film (#high texture film diamond L-B, Okura Industrial) sandwiched between PP mesh sheets were gently stirred in 200 mL of 1% PVP-I ethanol solution at room temperature; after 15 min, these materials were removed from the solution and dipped in 3 L of water for 3 min. This procedure was repeated with 3 L of purified water. After blotting the water with a paper towel, the materials were air-dried and evaluated for their color tones before and after integration with PVP-I (Table 2). These materials were then weighed, and their iodine content was determined by back-titration using a sodium thiosulfate solution, as presented in Table 2.

Table 2. Comparison of Ability and Capacity of Different Materials in Integration with PVP-I

Material (abbreviation and manufacturer)	Presence (+) or absence (−) of PVP-I-integration	Iodine weight per area of PVP-I-integrated materials (µg/cm2)
Polyester nonwoven fabric (NWF) (#DM105A, Kurashiki Textile Manufacturing, Osaka, Japan)	−	N.D.
Rayon NWF (#EWK-3043, Japan Vilene Company, Tokyo, Japan)	−	N.D.
Nylon-66 NWF (#PO303WTO, UNITIKA, Tokyo, Japan)	+	159.0
Polyolefin NWF (#DM35KW, SHINWA, Ehime, Japan)	−	N.D.
Polypropylene NWF (#6620-1A, SHINWA)	−	N.D.
Polyethylene woven fabric (WF) (#KIN-KG60777, WAVOU, Tokyo, Japan)	−	N.D.
Nylon-66 WF (#SMD-FR30, WAVOU)	+	121.0
Polyester WF (#T3010, UTAX, Hyogo, Japan)	−	N.D.
Cotton WF (#11WSR-456, WAVOU)	−	N.D.
Rayon (#9060, Inami, Tokyo, Japan)	−	N.D.
Nylon-66 film (#NO-25, UNITIKA)	+	127.0
Polyurethane film (#ET85, Okura Industrial, Kagawa, Japan)	+	70.0
Polyethylene terephthalate film (#vurex film A31, TEIJIN LIMITED, Osaka, Japan)	−	N.D.
Polyethylene film (#high texture film diamond L-B, Okura Industrial)	−	N.D.

N.D.: not detected.

Preparation of Nylon-66 and Polyurethane Materials Complexes Integrated with Different Concentrations of PVP-I

Nylon-66 NWF (UNITIKA) sandwiched between PP mesh sheets was gently stirred in 500 mL of 5% PVP-I ethanol solution at room temperature; after 10 min, the NWF was removed from solution and dipped in 3 L of water for 10 s. This procedure was repeated with 3 L of purified water. After blotting the water and air drying, the NWF was weighted (0.24 g). The iodine content, determined by back titration with sodium thiosulfate solution, was 53.9 µg/cm² (indicated as NWF-2 in Table 2). NWF-1 and -3 and nylon-66 WF (TOYOSHIMA TEXTILE, Fukui, Japan)-1, -2, and -3 were prepared in the same manner, as described in Table 2. NF (UNITIKA) was placed in 100 mL of 20% PVP-I ethanol solution and gently stirred at room temperature. After 120 min, the film was removed from the solution and washed with 3 L of purified water for 10 s. This procedure was repeated with 3 L of purified water. After air drying, the film was weighed (0.28 g). The iodine concentration, determined by back titration with sodium thiosulfate solution, was 120.24 µg/cm² (indicated as NF-3). NF-1 and -2 and PF (Okura Industrial)-1, -2, and -3 were prepared in the same manner, as described in Table 3.

Table 3. Conditions for Preparing PVP-I-Integrated Materials

Material (abbreviation and manufacturer)	Concentration of PVP-I in an ethanol solution (%)	Reaction time (min)	Material number and iodine weight per area of PVP-I-integrated material (µg/cm2)
Nylon-66 nonwoven fabrics (NWF) (#PO303WTO, UNITIKA, Tokyo, Japan)	1	6	NWF-1 22
	5	10	NWF-2 53.9
	5	30	NWF-3 99.8
Nylon-66 woven fabric (WF) (#SMD-FR30, WAVOU, Tokyo, Japan)	1	15	WF-1 24
	1)		WF-2 57.9
	20	90	WF-3 104.2
Nylon-66 film (NF) (#NO-25, UNITIKA, Tokyo, Japan)	2)		NF-1 25.8
	3)		NF-2 40
	20	120	NF-3 120.2
Polyurethane film (PF) (#ET85, Okura Industrial, Kagawa, Japan)	5	5	PF-1 23.9
	5	15	PF-2 59.7
	20	30	PF-3 119.8

1) Dip in 1% PVP-I solution for 30 min and then dip in 5% PVP-I solution for 10 min. Each step is performed twice. 2) Dip in 0.5% PVP-I solution for 3 min and then dip in 5% PVP-I solution for 30 min. Each step is performed twice. 3) Dip in 0.5% PVP-I solution for 10 min and then dip in 20% PVP-I solution for 30 min. Each step is performed twice.

Culture of MDCK Cells

MDCK cells (Cell Bank, Ibaraki, Japan) were cultured in a growth medium (high-glucose Dulbecco’s modified Eagle’s medium [DMEM; WAKO, Osaka, Japan], supplemented with 10% fetal bovine serum [FBS; Thermo Fisher Scientific, MA, U.S.A.], 100 units/mL penicillin and 100 µg/mL streptomycin [P/S; Thermo Fisher Scientific], and 4 mM L-glutamine) at 37 °C under 5% CO₂.

Strains of Influenza A Virus

Puerto Rico 8/34 (A/PR/8/34; H1N1), Wisconsin 33 (A/WSN/33; H1N1), and Aichi 2/68 (A/Aichi/2/68; H3N2) strains of the influenza A viruses were used for the experiments. Viral titers were determined by immunostaining of the influenza A viral nucleoprotein, as previously described.^16–18)

Evaluation of Prevention of Influenza A Virus Infection of MDCK Cells by PVP-I-Integrated Materials Using NB Staining and MTT Assay

MDCK cells were seeded in 96-well plates (1 × 10⁴ cells/well). One square centimeter of PVP-I-integrated material was placed in a 24-well plate. Two hundred microliters of growth medium with or without influenza A viruses at 20 multiplicity of infection (MOI) of A/PR/8/34 (H1N1), 0.2 or 2 MOI of A/WSN/33 (H1N1), and 2 or 20 MOI of A/Aichi/2/68 (H3N2) was then added to the wells. Wells with no material (control; Ctrl) Wells with materials not treated with PVP-I (blank) were used as negative controls. Each medium was incubated for 0.5 h at 37 °C in the presence of 5% CO₂, and 100 µL of each medium with or without influenza A viruses at 10 MOI of A/PR/8/34 (H1N1), 0.1 or 1 MOI of A/WSN/33 (H1N1), and 1 or 10 MOI of A/Aichi/2/68 (H3N2) was subsequently added to the wells containing MDCK cells in 96-well plates. After incubation of the PVP-I material, the Ctrl- or blank-treated virus media on the cell surface incubated for 72 h at 37 °C in the presence of 5% CO₂ were used to determine the viability of MDCK cells in each well through NB staining and MTT assay. NB staining was conducted as described previously,¹⁹⁾ and viable cells in each well were stained blue, whereas dead cells were unstained. The MTT assay was conducted using an MTT cell count kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions.

Measurement of the Amount of Iodine (I₂) in the Medium Eluted from PVP-I-Integrated Materials

Four pieces of NWF-1, WF-1, NF-1, and PF-1 with an area of 1 cm², respectively, were placed in a 24-well plate. Eight hundred microliters of FluorBrite™ DMEM (Thermo Fisher Scientific), phenol red-free high-glucose DMEM, supplemented with 4 mM of L-glutamine were then added to the wells, and each well was prepared as six wells. Wells with untreated materials (blank) were used as negative controls. Each well was incubated for 1 h at 37 °C in the presence of 5% CO₂ with stirring every 10 min. Four milliliters of each medium were collected and diluted to 10 mL with ultrapure water (diluted ×2.5). Powdered iodine reagent (Hanna Instruments, RI, U.S.A.) containing N,N-diethyl-p-phenylenediamine (DPD) was added to the diluted samples. The amount of iodine (I₂) in the samples was determined by the DPD method using a multiparameter photometer (HI83300-01, Hanna Instruments) according to the manufacturer’s instructions.

Statistical Analyses

The results of MTT assay were expressed as means ± standard error of the mean (S.E.M.). The statistical significance of the differences between more than two groups was analyzed using one-way ANOVA with the post hoc Tukey’s test. Differences were considered statistically significant at p < 0.05.

RESULTS

PVP-I Nylon-66 NWF Was Effective against Influenza A Virus Infection in MDCK Cells

To determine the concentration of PVP-I with effective anti-influenza virus activity for integration in nylon-66 NWF, various concentrations of PVP-I solution were used to integrate the nylon-66 NWF at various reaction times as indicated in Table 1. Back titration with sodium thiosulfate solution confirmed the presence of iodine on the nylon-66 NWF at concentrations of 10–30 and 50–100 µg/cm² when 1 and 5% PVP-I were used for integrating the NWF, respectively. The colors of nylon-66 NWF samples varied from white (blank) to dark yellow (Fig. 1A), with different PVP-I concentrations and reaction times (Table 1). The concentration of I₂ per 1 cm² of the PVP-I-integrated nylon-66 NWF was not constant. As the structure of NWF materials is not constant, the concentration of PVP-I fluctuated.

Fig. 1. Experimental Procedure for Studying the Protective Effect of PVP-I-Integrated Materials against Influenza A Infection in MDCK Cells

(A) Image showing 1-cm squares of nylon-66 NWF with or without PVP-I in a 24-well plate. NWFs were integrated with 10–30 or 50–100 µg/cm² of iodine as described in Table 1; well with no-NWF (Ctrl.) and NWF not treated with PVP-I (blank-NWF) were used as negative controls. (B) Experimental procedure for studying protection of MDCK cells from influenza A virus infection by PVP-I-integrated materials. One-cm squares of PVP-I materials were placed in a 24-well plate; subsequently, the growth medium with or without influenza A viruses, A/PR/8/34 (H1N1), A/WSN/33 (H1N1), or A/Aichi/2/68 (H3N2) was added to the wells. After 0.5 h of treatment, this medium was transferred onto MDCK cell surface. After 72 h of incubation, cell viability was determined via naphthol blue-black (NB) staining and thiazolyl blue tetrazolium bromide (MTT) assay.

To evaluate the anti-influenza activity of nylon-66 NWF integrated with PVP-I, the effect of the PVP-I integrating on the nylon-66 NWF on the survival of MDCK cells in the culture inoculated with influenza A viruses was examined according to the procedure shown in Fig. 1B. The growth mediums with or without influenza A viruses, A/PR/8/34 (H1N1), A/WSN/33 (H1N1), or A/Aichi/2/68 (H3N2) were placed on 1 cm² nylon-66 NWFs integrated with various concentrations of PVP-I (Fig. 1A, Table 1) and subsequently transferred to well containing the MDCK cells (Fig. 1B). The Ctrl. and blank-nylon-66 NWF were used as negative controls. MDCK cell viability in each well was determined via NB staining and MTT assay. The cells in the wells with no addition of viruses stained blue on NB staining (Figs. 2A, C, E, left panels); in the MTT assay, they showed similar levels of survival (Figs. 2B, D, F) at all iodine concentrations, suggesting that the concentrations used in this experiment did not induce toxicity in the MDCK cells. NB staining and MTT assay revealed that PVP-I nylon-66 NWF significantly retained the number of cells that survived the exposure to A/PR/8/34 (H1N1) (Figs. 2A, B), A/WSN/33 (H1N1) (Figs. 2C, D), or A/Aichi/2/68 (H3N2) (Figs. 2E, F) in a concentration-dependent manner (*** p < 0.001). Therefore, these results showed that the exposure of influenza A virus H1N1 and H3N2 strains to PVP-I nylon-66 NWF protected the MDCK cells from being infected.

Fig. 2. PVP-I Nylon-66 NWF Prevents MDCK Cells from Infection with Influenza A Viruses

Influenza A viruses, 0 or 10 MOI of A/PR/8/34 (H1N1) (A, B); 0, 0.1, or 1 MOI of A/WSN/33 (H1N1) (B, C); and 0, 1, or 10 MOI of A/Aichi/2/68 (H3N2) (E, F), exposed to 10–30 or 50–100 µg/cm². PVP-I-integrated nylon-66 NWF samples were added to wells containing MDCK cells, as shown in Fig. 1B. Wells with Ctrl. and blank-NWF were used as negative controls. MDCK cell survival rates 72 h after inoculation with viruses exposed to Ctrl., blank-NWF, and PVP-I-integrated nylon-66 NWF were determined using NB staining (A, C, E) and MTT assay (n = 9 each) (B, D, F). Data of NB staining are representative of three independent experiments (A, C, E). Data of MTT assay are represented as mean ± S.E.M. of three independent experiments (B, D, F). w/o; without. *** p < 0.001, versus the indicated groups. N.S.: not significant, versus the indicated groups.

Nylon-66 and Polyurethane Materials Were Integrated with PVP-I

To evaluate whether materials different from nylon-66 NWF could be integrated with PVP-I, we examined the integration of PVP-I into various materials, as shown in Fig. 3A. The colors of nylon-66 NWF, nylon-66 WF, NF, and PF varied from white or clear (Fig. 3A) to dark yellow (Fig. 3B). The presence of iodine in these materials was confirmed by back titration with a sodium thiosulfate solution when 1% PVP-I was used for integration (Table 2). These results indicated that nylon-66 and polyurethane materials, but not polyester, rayon, polyolefin, PP, PE, cotton, or PE terephthalate, can be integrated with PVP-I.

Fig. 3. Comparison of Various Materials after Integration of PVP-I

Images of various materials (A) before or (B) after integration with PVP-I in Table 2. (C) Images of 1 cm² pieces of materials, including nylon-66 NWF, nylon-66 woven fabric (WF), nylon-66 film (NF), and polyurethane film (PF), that were integrated with PVP-I at different concentrations (numbered 1, 2, and 3 in Table 3). Materials not treated with PVP-I (blank) were used as negative controls.

PVP-I Nylon-66 and Polyurethane Materials Were Effective in Preventing Influenza A Virus Infection

Subsequently, we evaluated the anti-influenza activities of PVP-I and nylon-66 or polyurethane complexes, namely NWF, WF, NF, and PF, integrated with PVP-I. These materials were combined with various concentrations of PVP-I (Table 3). Figure 3C shows the features of the different materials integrated with various PVP-I concentrations.

Their effect on the survival of MDCK cells in the culture inoculated with influenza A viruses, A/PR/8/34 (H1N1), A/WSN/33 (H1N1), or A/Aichi/2/68 (H3N2) was examined according to the procedure shown in Fig. 1B. Wells containing influenza A viruses exposed to the blank-NWF showed lower cell survival than those containing the medium exposed to the blank-NWF on NB staining (Figs. 4A, C, E) and in the MTT assay (Figs. 4B, D, F). Not only the wells inoculated with viruses exposed to the blank-NWF but also the wells inoculated with viruses exposed to the other blank materials showed a significant reduction in cell survival (*** p < 0.001) (Figs. 4B, D, F), indicating that blank-NWF, -WF, -NF, and -PF without PVP-I did not protect the MDCK cells from the virus. No significant difference in cell survival was observed when Ctrl. or blank-NWF was tested (Fig. 2) and when blank-NWF, -WF, -NF, or -PF was tested (Figs. 4B, D, F), suggesting that these materials have no effect on cell survival.

Fig. 4. Comparison of Efficacy of Different Materials Integrated with PVP-I in Preventing Cells from Being Infected with Influenza A Viruses

The growth medium with or without influenza A viruses, 10 MOI of A/PR/8/34 (H1N1) (A, B), 0.1 MOI of A/WSN/33 (H1N1) (B, C), and 1 MOI of A/Aichi/2/68 (H3N2) (E, F), incubated with PVP-I nylon-66 NWF-, nylon-66 WF-, NF-, or PF-1–3 (Fig. 3C, Table 3) in 24-well plate were transferred to well containing MDCK cells, as shown in Fig. 1B. Each material not treated with PVP-I (blank) was used as a negative control. MDCK cell viability was determined via NB staining (A, C, E) and MTT assay (B, D, F) after 72 h of incubation. Data of NB staining are representative of three independent experiments (A, C, E). Data of MTT assay are represented as mean ± S.E.M. of three independent experiments (B; n = 9 each, D; n = 12 each, and F; n = 9 each). ** p < 0.01, *** p < 0.001, versus blank-NWF, WF, NF, or PF without viruses. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001, versus blank-NWF, WF, NF, or PF with viruses. ^‡‡‡p < 0.001, versus the indicated groups for NWF-1 with viruses. ^§p < 0.05, ^§§p < 0.01, ^§§§p < 0.001, versus the indicated groups for NWF-2 with viruses. N.S. (not significant), versus the indicated groups.

In the absence of the virus, significant reduction of cell survival was observed on NB staining (Figs. 4A, C, E) and in the MTT assay (*** p < 0.001) (Figs. 4B, D, F) when NWF-3, NF-3, and PF-3 were tested in comparison with their own blanks. These results suggest that the high concentrations of PVP-I integrating on NWF-3, NF-3, and PF-3 induced cell toxicity, whereas low PVP-I concentrations did not induce toxicity (Fig. 4).

In the presence of the virus at non-cytotoxic concentrations of PVP-I, significantly high levels of cell survival were observed when the PVP-I-integrated materials were tested with NB staining (Figs. 4A, C, E) and MTT assay (^†p < 0.05, ^††p < 0.01, and ^†††p < 0.001; Figs. 4B, D, F) in comparison with their own blanks (NWF, WF, NF, and PF). Among the material used, NWF-1 and NWF-2 showed better cell survival in the presence of A/PR/8/34 (H1N1) and A/WSN/33 (H1N1) than the other materials integrated with similar concentrations of PVP-I (^‡‡‡p < 0.001 and ^§p < 0.05, ^§§p < 0.01, and ^§§§p < 0.001; Fig. 4), respectively.

As NWF-1 significantly increased the survival of cells infected with A/PR/8/34 (H1N1) and A/WSN/33 (H1N1) viruses compared with WF-1, NF-1, and PF-1 (Figs. 4A–D), we evaluated the differences in the amount of eluted PVP-I. The amount of I₂ in the medium was measured using the DPD method. I₂ eluted from NWF-1, WF-1, NF-1, and PF-1 was detected compared with their respective blanks, and NWF-1 had a higher amount of eluted I₂ in the medium than the other materials, although no significant difference was observed among them (Fig. 5).

Fig. 5. Determination of Iodine (I₂) Eluted from PVP-I-Integrated Materials

NWF-1, WF-1, NF-1, and PF-1 with an area of 1 cm², as presented in Table 3, were placed in a 24-well plate, respectively. FluorBrite™ DMEM, a phenol red-free high-glucose DMEM, was then added to the wells. Wells with materials not treated with PVP-I (blank) were used as negative controls. Each well was incubated for 1 h at 37 °C, and powdered iodine reagent containing N,N-diethyl-p-phenylenediamine (DPD) was added to the collected samples. The amount of iodine (I₂) in the medium in blank (n = 4 each) or PVP-I-integrated materials (n = 4 each) was determined by the DPD method using a multiparameter photometer. Data are represented as mean ± S.E.M. of four independent experiments. N.D.: not detected.

Overall, these results indicated that PVP-I materials used in this study were effective against influenza A infection and that nylon-66 NWF is the best material for integration with PVP-I to ensure anti-influenza activity. The amount of PVP-I eluted from each material would show differences in anti-influenza activity.

DISCUSSION

In this study, we integrated PVP-I with nylon-66 NWF and evaluated its antiviral activity against influenza A viruses by examining the viability of MDCK cells after infection. The PVP-I nylon-66 NWF protected the MDCK cells from infection in a PVP-I concentration-dependent manner. We integrated PVP-I into various materials, such as nylon-66 and polyurethane. These PVP-I materials were also effective against influenza virus infection, and treatment with PVP-I nylon-66 NWF showed the highest cell survival among all the tested materials. PVP-I showed anti-influenza A virus activity when used in conjunction with PPE materials. Moreover, nylon-66 NWF integrated with PVP-I was found to be the best material to ensure anti-influenza activity.

Face masks and respirators (e.g., surgical mask and N95 respirator) generally consist of three layers: outer, middle, and inner layers. The middle filter layer is an important layer that protects the wearer against invasive particles and infective droplets and aerosols containing bacteria/viruses. This filter layer is usually made of melt-blown NWF, fabricated from synthetic or natural polymers composites, such as PP, PE, or nylon.⁴⁾ However, medical masks composed of NWF filter layers only capture the particles and droplets and cannot kill or inactivate the infective organisms. To address this problem, we prepared a nylon-66 NWF filter layer integrated with PVP-I. We found that the PVP-I-integrated NWF samples protected the MDCK cells from influenza A viruses, A/PR/8/34 (H1N1), A/WSN/33 (H1N1), or A/Aichi/2/68 (H3N2), suggesting that the PVP-I integrated into the NWF can kill or inactivate influenza A viruses. PVP-I has anti-viral activities against enveloped viruses, such as influenza virus,^9,10) Middle East respiratory syndrome-related coronavirus,²⁰⁾ SARS-CoV-2,^11–13) Ebola virus,²¹⁾ and human immunodeficiency virus,²²⁾ and nonenveloped viruses.⁷⁾ Thus, PVP-I nylon-66 NWF could be used to develop more effective anti-viral masks than the existing ones.

The usefulness of multi-functional masks with filter layers integrated with anti-bacterial and anti-viral components has been reported previously.⁴⁾ For anti-viral efficiency, various anti-viral components, such as metal-based nanoparticles of Ag, Au, Cu, CuO, TiO₂, ZnO, or SiO₂-Ag; licorice root extract; salt; and antibodies have been integrated into filter layer materials.⁴⁾ PVP-I has been widely used as an antiseptic solution for more than 60 years given its potent broad-spectrum activity against bacteria, virus, fungi, and protozoa^5,6) and attractive properties including anti-corrosion, established safety, lack of resistance, accessibility, and low cost.²³⁾ PVP-I integrates into a filter layer and hence is used for developing multi-functional masks with anti-bacterial and viral activities, and our method of integrating nylon-66 NWF with PVP-I could be applied to it. Additionally, it is important to wear not only face masks or respirators but also other PPEs, such as gloves, hats, earplugs, vests, and body suits, to prevent infections from pathogens in healthcare settings. These PPE products are made from various materials including nylon and polyurethane.²⁴⁾ For example, nylon material is used to produce gloves, headbands, ear plugs, vests, and bodysuits, while polyurethane is used as a buffer layer in safety helmets, which is made of deformable paddings.²⁴⁾ In present study, we found that nylon-66 and polyurethane materials, but not polyester, rayon, polyolefin, PP, PE, cotton, and PE terephthalate, could be integrated with PVP-I (Fig. 3B, Table 2). We integrated nylon-66 NWF, nylon-66 WF, NF, and PF with various concentrations of PVP-I (Fig. 3C, Table 3) and found that they were effective in preventing influenza A viral infection. Thus, nylon-66 and polyurethane materials integrated with PVP-I can be used to develop multifunctional PPEs with antibacterial and antiviral properties.

Among the four different materials, PVP-I nylon-66 NWF evoked the highest cell survival rate at a significant level (Figs. 4A–D). In general, NWF has a higher elongation, flow rate, permeability, and dielectric permittivity, and smaller pores than other materials, allowing it to easily capture droplets and aerosols.^4,25–27) Drug-integrated NWF can also facilitate drug dissolution into captured droplets and aerosols.²⁸⁾ We showed that I₂ eluted from NWF-1, WF-1, NF-1, and PF-1 was detectable compared with their respective blanks, and NWF-1 had a higher amount of eluted I₂ in the medium than the other materials (Fig. 5). Thus, a higher amount of PVP-I was eluted from PVP-I nylon-66 NWF than from the other materials when the virus medium was added. The PVP-I eluted from each material killed or inactivated the influenza viruses.

Although nylon-66 NWF, nylon-66 WF, NF, and PF could be integrated with PVP-I, polyester, rayon, polyolefin, PP, PE, cotton, and PE terephthalate materials could not be integrated with it (Figs. 3A, B, Table 2). The PVP-I·nylon-66 complex is believed to be bound by some form of intermolecular forces, such as ionic bonding or hydrogen bonding, between the protons of PVP-I and the amide groups of nylon-66 as PE does not have amide or carbamate groups in its chemical structure that can react with iodine protons. The mechanism of PVP-I·polyurethane complex formation is assumed to be similar. Figure 6 shows the binding through intermolecular forces formed between PVP-I and the amide group of nylon-66 (Fig. 6A) or the carbamate group of polyurethane (Fig. 6B).

Fig. 6. Proposed Formation of Integrated PVP-I·Nylon-66 or Polyurethane Complexes

Proposed structures of (A) PVP-I·nylon-66 or (B) polyurethane complexes that are potentially formed in the PVP-I-integrated materials.

In conclusion, the integrated PVP-I nylon-66 NWF material protected the MDCK cells from influenza virus infection. We integrated PVP-I into other materials, such as nylon-66 and polyurethane. These PVP-I materials were also effective against influenza virus infection, and treatment with PVP-I nylon-66 NWF showed the highest cell survival rate among all the tested materials. This indicated that nylon-66 NWF integrated with PVP-I is the best material to ensure anti-influenza activity. Therefore, PVP-I-integrated masks could have the potential to inhibit respiratory virus infections. Our results provide new information for developing multi-functional PPEs with anti-viral activity by integrating them with PVP-I to prevent the transmission of respiratory viruses.

Acknowledgments

This work was supported by a Grant from Tokushima Bunri University and partly by funding from DIA Pharmaceutical Co., Ltd.

Conflict of Interest

MS, KI, NS, ET, HK, YS, KT and TK declare no conflict of interest. YS is a technical advisor of DIA Pharmaceutical Co., Ltd. HM is an employee of DIA Pharmaceutical Co., Ltd., and SM is the employer of DIA Pharmaceutical Co., Ltd.

REFERENCES

• 1) Cowling BJ, Ip DK, Fang VJ, Suntarattiwong P, Olsen SJ, Levy J, Uyeki TM, Leung GM, Malik Peiris JS, Chotpitayasunondh T, Nishiura H, Mark Simmerman J. Aerosol transmission is an important mode of influenza A virus spread. Nat. Commun., 4, 1935 (2013).
• 2) Liu Y, Ning Z, Chen Y, Guo M, Liu Y, Gali NK, Sun L, Duan Y, Cai J, Westerdahl D, Liu X, Xu K, Ho KF, Kan H, Fu Q, Lan K. Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals. Nature, 582, 557–560 (2020).
• 3) Ueki H, Furusawa Y, Iwatsuki-Horimoto K, Imai M, Kabata H, Nishimura H, Kawaoka Y. Effectiveness of face masks in preventing airborne transmission of SARS-CoV-2. mSphere, 5, e00637-20 (2020).
• 4) Deng W, Sun Y, Yao X, Subramanian K, Ling C, Wang H, Chopra SS, Xu BB, Wang JX, Chen JF, Wang D, Amancio H, Pramana S, Ye R, Wang S. Masks for COVID-19. Adv. Sci. (Weinh.), 9, 2102189 (2022).
• 5) Barreto R, Barrois B, Lambert J, Malhotra-Kumar S, Santos-Fernandes V, Monstrey S. Addressing the challenges in antisepsis: focus on povidone iodine. Int. J. Antimicrob. Agents, 56, 106064 (2020).
• 6) Zamora JL. Chemical and microbiologic characteristics and toxicity of povidone-iodine solutions. Am. J. Surg., 151, 400–406 (1986).
• 7) Eggers M. Infectious disease management and control with povidone iodine. Infect. Dis. Ther., 8, 581–593 (2019).
• 8) Kawana R, Kitamura T, Nakagomi O, Matsumoto I, Arita M, Yoshihara N, Yanagi K, Yamada A, Morita O, Yoshida Y, Furuya Y, Chiba S. Inactivation of human viruses by povidone-iodine in comparison with other antiseptics. Dermatology, 195 (Suppl. 2), 29–35 (1997).
• 9) Ito H, Ito T, Hikida M, Yashiro J, Otsuka A, Kida H, Otsuki K. Outbreak of highly pathogenic avian influenza in Japan and anti-influenza virus activity of povidone-iodine products. Dermatology, 212 (Suppl. 1), 115–118 (2006).
• 10) Sriwilaijaroen N, Wilairat P, Hiramatsu H, Takahashi T, Suzuki T, Ito M, Ito Y, Tashiro M, Suzuki Y. Mechanisms of the action of povidone-iodine against human and avian influenza A viruses: its effects on hemagglutination and sialidase activities. Virol. J., 6, 124 (2009).
• 11) Anderson DE, Sivalingam V, Kang AEZ, Ananthanarayanan A, Arumugam H, Jenkins TM, Hadjiat Y, Eggers M. Povidone-iodine demonstrates rapid in vitro virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease. Infect. Dis. Ther., 9, 669–675 (2020).
• 12) Wang Y, Wu Y, Wang Q, Zhu J, Shi W, Han Z, Zhang Y, Chen K. Virucidal effect of povidone-iodine against SARS-CoV-2 in vitro. J. Int. Med. Res., 49, 3000605211063695 (2021).
• 13) Shet M, Hong R, Igo D, Cataldo M, Bhaskar S. In vitro evaluation of the virucidal activity of different povidone-iodine formulations against murine and human coronaviruses. Infect. Dis. Ther., 10, 2777–2790 (2021).
• 14) Zarabanda D, Vukkadala N, Phillips KM, Qian ZJ, Mfuh KO, Hatter MJ, Lee IT, Rao VK, Hwang PH, Domb G, Patel ZM, Pinsky BA, Nayak JV. The effect of povidone-iodine nasal spray on nasopharyngeal SARS-CoV-2 viral load: a randomized control trial. Laryngoscope, 132, 2089–2095 (2022).
• 15) Matsuyama A, Okura H, Hashimoto S, Tanaka T. A prospective, randomized, open-label trial of early versus late povidone-iodine gargling in patients with COVID-19. Sci. Rep., 12, 20449 (2022).
• 16) Shoji M, Arakaki Y, Esumi T, Kohnomi S, Yamamoto C, Suzuki Y, Takahashi E, Konishi S, Kido H, Kuzuhara T. Bakuchiol is a phenolic isoprenoid with novel enantiomer-selective anti-influenza A virus activity involving Nrf2 activation. J. Biol. Chem., 290, 28001–28017 (2015).
• 17) Shoji M, Sugimoto M, Matsuno K, Fujita Y, Mii T, Ayaki S, Takeuchi M, Yamaji S, Tanaka N, Takahashi E, Noda T, Kido H, Tokuyama T, Tokuyama T, Tokuyama T, Kuzuhara T. A novel aqueous extract from rice fermented with Aspergillus oryzae and Saccharomyces cerevisiae possesses an anti-influenza A virus activity. PLOS ONE, 16, e0244885 (2021).
• 18) Shoji M, Esumi T, Tanaka N, Takeuchi M, Yamaji S, Watanabe M, Takahashi E, Kido H, Yamamoto M, Kuzuhara T. Organic synthesis and anti-influenza A virus activity of cyclobakuchiols A, B, C, and D. PLOS ONE, 16, e0248960 (2021).
• 19) Shoji M, Takahashi E, Hatakeyama D, Iwai Y, Morita Y, Shirayama R, Echigo N, Kido H, Nakamura S, Mashino T, Okutani T, Kuzuhara T. Anti-influenza activity of c60 fullerene derivatives. PLOS ONE, 8, e66337 (2013).
• 20) Eggers M, Eickmann M, Zorn J. Rapid and effective virucidal activity of povidone-iodine products against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Modified Vaccinia Virus Ankara (MVA). Infect. Dis. Ther., 4, 491–501 (2015).
• 21) Eggers M, Eickmann M, Kowalski K, Zorn J, Reimer K. Povidone-iodine hand wash and hand rub products demonstrated excellent in vitro virucidal efficacy against Ebola virus and modified vaccinia virus Ankara, the new European test virus for enveloped viruses. BMC Infect. Dis., 15, 375 (2015).
• 22) Sabracos L, Romanou S, Dontas I, Coulocheri S, Ploumidou K, Perrea D. The in vitro effective antiviral action of povidone-iodine (PVP-I) may also have therapeutic potential by its intravenous administration diluted with Ringer’s solution. Med. Hypotheses, 68, 272–274 (2007).
• 23) Dang T, Yim N, Tummala S, Parsa AA, Parsa FD. Povidone-Iodine versus antibiotic irrigation in breast implant surgery: revival of the ideal solution. J. Plast. Reconstr. Aesthet. Surg., 73, 391–407 (2020).
• 24) Shi J, Li H, Xu F, Tao X. Materials in advanced design of personal protective equipment: a review. Mater. Today Adv., 12, 100171 (2021).
• 25) Borojeni IA, Gajewski G, Riahi RA. Application of electrospun nonwoven fibers in air filters. Fibers, 10, 15 (2022).
• 26) Hassan MA, Yeom BY, Wilkie A, Pourdeyhimi B, Khan SA. Fabrication of nanofiber meltblown membranes and their filtration properties. J. Memb. Sci., 427, 336–344 (2013).
• 27) Bi S, Tang J, Wang D, Su Z, Hou G, Li H, Li J. Lightweight non-woven fabric graphene aerogel composite matrices for assembling carbonyl iron as flexible microwave absorbing textiles. J. Mater. Sci. Mater. Electron., 30, 17137–17144 (2019).
• 28) Rostamitabar M, Abdelgawad AM, Jockenhoevel S, Ghazanfari S. Drug-eluting medical textiles: from fiber production and textile fabrication to drug loading and delivery. Macromol. Biosci., 21, 2100021 (2021).