Zinc Oxide Nanoparticle-Loaded Electrospun Polyvinylidene Fluoride Nanofibers as a Potential Face Protector against Respiratory Viral Infections.

ZnO-NPs loaded polyvinylidene fluoride (PVDF) composite nanofibers were fabricated by electrospinning and optimized using different concentrations (0, 2, and 5 wt %) of ZnO-NPs. Characterization techniques, for example, FTIR, SEM, XRD, and tensile strength analysis were performed to analyze the composite nanofibers. Molecular docking calculations were performed to evaluate the binding affinity of PVDF and ZnO@PVDF against the hexon protein of adenovirus (PDB ID: 6CGV). The cytotoxicity of tested materials was evaluated using MTT assay, and nontoxic doses subjected to antiviral evaluation against human adenovirus type-5 as a human respiratory model were analyzed using quantitative polymerase chain reaction assay. IC50 values were obtained at concentrations of 0, 2, and 5% of ZnO-loaded PVDF; however, no cytotoxic effect was detected for the nanofibers. In 5% ZnO-loaded PVDF nanofibers, both the viral entry and its replication were inhibited in both the adsorption and virucidal antiviral mechanisms, making it a potent antiviral filter/mask. Therefore, ZnO-loaded PVDF nanofiber is a potentially prototyped filter embedded in a commercial face mask for use as an antiviral mask with a pronounced potential to reduce the spreading of infectious respiratory diseases, for example, COVID-19 and its analogues.

Introduction

Preventing the spread of the novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), publicly known as COVID-19, became a worldwide demand to overcome the current global crisis with its several mutations such as Omicron and allow people to get back to their normal lives. The origin of coronavirus family is known as Coronaviridae, which also includes genus Torovirus. These two genera share a very similar structure.[1] SARS-CoV-2 is a single-stranded RNA virus that has a surface spike layer (20 nm).[2,3] The devastating strain of SARS-CoV-2 was reported first in Wuhan, China, in December 2019 and notified as a world pandemic by the World Health Organization (WHO) in March 2020.[4] It was reported that the pandemic resulted in around 5.5 million deaths resulting from more than 308 million global infections till 2022[5] (https://www.worldometers.info/coronavirus/, 2022.01.10). The infection transmits through inhalations via droplets and aerosols when infected people sneeze or cough; breeze; or by being in contact with infected surfaces.[6] The fine virus particles remain infectious for at least 16 h, and their inhaled aerosols infect the cells of the upper and lower respiratory tracks.[6] Subsequently, WHO suggested wearing surgical masks for human protection and to limit the spread of SARS-CoV-2 as well as other respiratory infectious diseases.[7] Recent studies show that facial protection via filtering face pieces and masks may reduce the spread of viruses such as influenza and Corona viruses by 85%.[6]

During the current pandemic, and generally during epidemics, there is a noticeable shortage in personal protective equipment, especially with the expectation of new upcoming infections’ waves and/or virus mutations. There is always extensive use of masks, gloves, protective suits, face shields, gowns, and N95-grade masks by healthcare workers and general public, which represents the frontline in reducing the infection risk.[8] Therefore, viral disinfection and reuse of personal protective materials might allow their reutilization. Examples of disinfection methods are heat,[9,10] ultraviolet (UV) irradiation,[11,12] steam,[13] ozone,[14] vaporized hydrogen peroxide,[15] chemical disinfectants,[12] and autoclaving.[16]

Different organic compounds and metal oxides exhibit antibacterial and antimicrobial activities.[17] Organic biocides such as triclosan, biguanides, chlorohexidine, and quaternary ammonium compounds were used in nanofiber fabrication to enhance the antimicrobial activity.[7]

In addition, a wide variety of metal/metal oxides of titanium (Ti), zinc (Zn), copper (Cu), and silver (Ag) have been examined in soluble and insoluble forms due to their small sizes and high surface area. They can inactivate the viral particles through interfering with the surface antigen and blocking their attachment to the host cell receptors,[5] while the ions of Ti and Zn are adsorbed on the bacterial cell surface to inhibit its protein activity. Human protective materials have been developed in parallel to vaccine production.[18−20]

It is important to fabricate biocidal masks that are safe upon disposal without causing secondary infections. Therefore, focusing on the selection of biodegradable, breathable, and virucidal materials enables superior protection against SARS-CoV-2 and bacteria, and extending the application to other types of PPE materials like body protection gowns and textiles for hospital uses thus increases their benefits. Also, application of antiviral agents to fabricate masks is a strategic technology to overcome the transmission of SARS-Cov-2 and such innovative idea depends on used materials.[21,22]

Various mask technologies and advanced materials are examined to overcome the critical mask shortages, cross-infection, and secondary transmission risk. Enormous efforts have been directed to improve the mask performance by incorporating new functionalities, such as using metal nanoparticles and herbal extracts to inactivate pathogens, graphene to make masks photothermal and superhydrophobic, and triboelectric nanogenerators (TENGs) to extend the mask lifetime.[23] Recently, the immobilization of Ag NPs on cotton fabrics of about 2–4 nm and 10 nm was studied, and the cotton fabric was observed to exhibit antiviral activity against influenza A and feline calicivirus.[24] Highly durable, antibacterial, and UV protective cotton fabrics were developed by the in situ synthesis of ZnO-NPs using hexamethyltriethylene tetramine (HMTETA) without the support of capping or other stabilizing agents.[25] The nanofibers were designed with a side-by-side structure of ZnO-NPs on one side and Ag-NPs on the other side, thereby forming a coupled fiber with the properties of photocatalysis, excellent antibacterial activity against Gram-positive and Gram-negative bacteria, and enhanced filtration efficiency.[26] The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on Cu–zeolite-coated textiles, even after short incubation, due to the presence of Cu2+ ions, as compared to the zeolite-coated textiles.[27] CuO-NP-embedded nanofibers were fabricated with a hydrophobic polymer–polyvinylpyrrolidone (PVP) using electrospinning, and CuO nanoparticles were exposed from the PVP polymer surface by etching the nanofiber with oxygen plasma and tested against H1N1 virus using quantified real-time polymerase chain reaction (RT-qPCR). The antiviral efficacy of CuO nanoparticle-incorporated nanofibers and results revealed that 70% of viruses were inactivated after 4 h of contact.[28]

Here, the antiviral activity was tested against human adenovirus type-5 (ADV-5), which, in its structure, mimics respiratory viruses as it just needs the traditional facilities of BSL2. Adenoviruses are double-stranded DNA viruses having a genome around ∼36 kb long.[29] They are always associated with a wide range of human diseases, including respiratory tract infections and ocular and gastrointestinal tract disorders, predominantly affecting children and young adults.[30]

Recently, nanofibers are being intensively and significantly employed in several applications due to their ease of preparation, enhanced surface area, and the possibility to host some other materials like nanoparticles. One of these potential applications include antiviral materials and antiviral masks.[31] Nanofibers can be lab-manufactured via phase separation (sol–gel), chemical vapor deposition, conjugate spinning, self-assembly, melt blowing, and electrospinning.[32] Electrospinning forms 3D nanofibers that are characterized by a wide variety of pore diameter distribution, high porosity, and effective mechanical properties, which allow it to reach the industrial stage.[33] Moreover, they have excellent thermal/mechanical properties, while their chemical resistance can be improved by loading with different materials such as Ag, CuO, Al2O3, TiO2, and ZnO, with the ability to use spinnable biodegradable polymers such as PVDF and PAN, which might help in reducing the global waste disposal problem.[7,34] Furthermore, the in-situ growth of nanoparticles on PPE surfaces may lead to leaching at some point of usage, which may cause serious health problems or discomfort for end-users; the binder may also affect their properties; so, this problem was fixed via electrospinning technology that affords increasing the polymeric surface area and enhancing the stability of impeded nanomaterials on the nanofiber structure.[35−38]

For the interpretation of material interactions, molecular docking is among the most widely used molecular modeling methods. It is mainly used to model the binding sites and interactions within protein–protein or protein–ligand complexes at the atomistic level 1. In docking calculations, two steps are employed: sampling of different poses and the assessment of the binding affinity between molecular structures. There are many available packages that perform well in predicting the binding affinity of small molecules to the protein structure. AutoDock Vina is one of the most used docking packages in protein–ligand interactions 2.[39−41]

In this work, the pristine nanofiber of PVDF which has “no” antiviral activity against respiratory viral infections, such as adenovirus, has been successfully converted into potent antiviral material via introducing a low-cost metal oxide (ZnO-NPs) in its nanofiber matrix. For overcoming the current Coronavirus pandemic and infectious diseases, the antiviral material-loaded electrospun nanofibers were fabricated and demonstrated applications in potent personal protective tools, such as face masks, filters, and hospital gowns. Instead of the current commercially used polypropylene face masks, the implementation of commercial face masks such as N95 with degradable antiviral nanofiber filters (2 × 2 cm2), or the antiviral nanofiber itself as a face mask internal layer, shall aid in overcoming the current pandemic due to the high filtration ability and comfortability. ZnO-NPs at different concentrations of 0, 2, and 5% loaded electrospun PVDF NFs were successfully fabricated by electrospinning, with diameters of 194, 164, and 83 nm, respectively. In the cost-effective one-layer form with enhanced mechanical properties, the electrospinning conditions, it optimized and intensively characterized to ensure their successful preparation and encapsulation efficiency. The cytotoxicity of PVDF and ZnO-NPs loaded PVDF NFs in 2 and 5% was examined by MTT assay, and the antiviral activity of nanofibers was evaluated using quantitative real-time PCR assay.

Materials and Methods

Materials

Polyvinylidene fluoride (PVDF; MW, 270,000 g/mol) was purchased from Sigma-Aldrich, Germany. N,N-dimethyl formamide (DMF) of purity ≥99% and acetone (HPLC grade, ≥99.9%) were obtained from Fisher Chemical, Germany. Absolute ethanol (99.9%) and dimethyl sulfoxide (DMSO, freshly distilled) were obtained from Analysis Co., Egypt. Zinc acetate (purity ≥99%) and polyethylene glycol (PEG) were obtained from Sigma-Aldrich, Germany. Deionized water was obtained via a MilliQ filtration system. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) dye was purchased from Serva Electrophoresis GmbH, Germany. Phosphate-buffered saline (PBS) tablets (pH 7.4) were obtained from Loba Chemie, India, where each tablet was dissolved in 200 mL of DI H2O. QIAamp Viral RNA extraction kit was purchased from Qiagen, Valencia, USA. cDNA synthesis kit was obtained from Roche Diagnostics, Germany. RT-qPCR was performed using Power SYBR Green PCR Master Mix, which was obtained from Thermo Scientific, USA. Vero cell lines ATCC:CCL-81 developed from monkey kidney cells were purchased commercially from the Holding Company for Biological Products and Vaccines (VACSERA, Cairo).

In the current work, an in silico study was performed against the hexon protein of adenovirus; such protein was retrieved from the protein data bank (PDB, ID 6CGV), while the structures of PVDF and ZnO@PVDF composite nanofibers were built using Avogadro which were then subjected to minimize the calculation using the quantum chemistry program Orca. The AutoDock Tools suit was used to prepare ligand and protein pdbqt files, while the AutoSite software was used to predict the protein binding sites, where the binding pocket with the highest score was selected.[42,43] AutoDock Vina was used for docking, while the protein–ligand interaction profiler (PLIP) analysis was used to predict different interactions inside the binding pockets.

Fabrication of Electrospun ZnO-Loaded PVDF Composite Nanofibers

Synthesis of ZnO-NPs

ZnO nanoparticles were prepared by the hydrothermal method. In this process, zinc acetate was dissolved in water (0.5 M) and stirred for 2 h to obtain a clear and homogeneous solution. To avoid the agglomeration of nanoparticles, PEG was added to the resultant solution which was used as a surfactant and again stirred for another 1 h. The resulting solution was transferred to a Teflon-capped autoclave and kept at 140 °C for 15 min. After the reaction process, the autoclave was cooled down to room temperature. The obtained precipitate was washed with water and ethanol, and the whole process was repeated for the neat ZnO. Finally, the powders were dried at 80 °C in a hot air oven for 12 h and then annealed in a tube furnace at 400 °C for 2 h (Scheme . The resultant ZnO-NPs were obtained with the calculated average particle size of 25.5 (±4) nm (Figure S1).

Scheme 1

Preparation of ZnO-NPs, ZnO-NPs@PVDF Nanofibers, and the Antiviral Activity

Image created with BioRender.com.

Nanofiber Preparation

15% PVDF (w/v) was prepared as follows: 15 g of PVDF was dissolved well in a 100 mL of DMF/acetone mixture solvent (7:3), and the mixture was kept under stirring for 12 h at 70 °C. 0, 2, and 5% (w/w) of ZnO-NPs were loaded onto the PVDF solution (total volume from 5 mL PVDF solution); 15 mg and 37.5 mg of ZnO-NPs were loaded, respectively. The composite solution of ZnO-NPs was mixed very carefully with the PVDF solution and then ultrasonicated for 1 h to ensure the homogeneous disruption of ZnO-NPs before electrospinning.

Electrospinning was performed using a clip spinneret for 2 mL of solutions of blank PVDF (0 wt % of ZnO-NPs), 2% ZnO-NPs/PVDF, and 5% ZnO-NPs/PVDF composite nanofibers (Scheme ). The applied voltage range was 18–23 kV, distance was 14 cm, spinneret speed was 100 mm, spinneret width was 80 mm, and the feed rate was tested at 0.9, 1.0, and 1.8 mL/h, respectively.

Cytotoxicity and Antiviral Activity of ZnO-Loaded PVDF Composite Nanofibers

Cytotoxicity by MTT Assay

The cytotoxicity of the fabricated composite nanofibers was tested using MTT assay on Vero cell lines. Briefly, cell monolayers were treated with the tested nanofibers for 48 h and then incubated with MTT solution into 5 mg/mL PBS till the formation of formazan crystals, which was visually confirmed using phase contrast microscopy. DMSO (100 μL/well) was added to dissolve the formazan crystals with shaking for 10 min. The absorbance was measured at 570 nm against blank (media only) on a microplate reader model (BMG Technologies, Germany) according to previously published protocols.[44,45] The antiviral activity of ZnO-loaded PVDF composite nanofibers against ADV-5 (viral model) was measured using RT-qPCR assay.

This step was performed typically as follows: (a) Infectivity assay: Human ADV-5 (ATCC VR-5) was propagated into Vero cells, and the viral load was determined using RT-qPCR assay,[9] (b) titration of ADV-5 DNA was performed after infecting Vero cells with a twofold serial dilution of ADV-5 stock of known viral load, followed by incubation for 48 h till 80–90% cell lysis; then, IC50 was identified by the viral dilution that affects 50% of cells using RT-qPCR assay; then, the known viral titer was used in the antiviral assay. (c) The antiviral activity of tested nanoparticles was evaluated against ADV-5 via two antiviral mechanisms as follows:[46]

Adsorption mechanism: A six-well plate was seeded with 5 × 105 cells/mL; after 24 h, it was treated with our materials for another 24 h, then infected with 100 IC50 of the previously detected viral copies/mL and then subjected to RT-qPCR, as previously described in determining the viral load in infectivity assay.

Virucidal mechanism: this method was performed to investigate the ability of the tested materials to neutralize the virus and then block its ability to infect and replicate into cells. A six-well plate was seeded with 5 × 105 cells/mL and then treated with our materials after its incubation with 100 IC50 of viral load at 4 °C for 1 h; then, incubation was continued for another 24 h under standard conditions. The plate was then subjected to RT-qPCR assay to measure the viral load.

Instrumental Characterization

The chemical structure of the fabricated nanofibers was analyzed using a FTIR spectrophotometer (Bruker Vertex 70, Germany), via the attenuated total reflection (ATR) method, and scanned from 4000 to 400 cm–1 with a scan rate of 2 cm–1 s–1. The crystal phases and crystallite size were detected by an X-ray diffractometer (Shimadzu 7000, Japan) with Cu Kα radiation (λ = 1.5418 Å), at 45 kV and 45 mA. The SEM, EDX, and mapping measurements were performed using a FE-SEM system (Quattro S from Thermo Fisher, USA) at an accelerating voltage of 5–20 kV. The zeta potential and particle size of nanoparticles were measured using a Zetasizer Nano Series instrument (Malvern Nano-ZS, England, UK).

The mechanical strength of different nanofibers was measured by a standard uniaxial tensile test (Z050, Zwick Roell AG, Ulm, Germany). The mechanical parameters such as maximum strength, elongation-to-break (%), and Young’s modulus were measured for all tested nanofibers. The cell viability of the tested materials was determined by a microplate reader (Clariostar Plus, BMG Labtech, Germany). The RT-qPCR assay was performed with Power SYBR Master Mix (Thermo Fisher Scientific, USA) using a PCR machine (Applied Biosystems 7500 system, Foster City, USA).

Results and Discussion

The electrospun materials were optimized to obtain nanofibers; the produced nanofibers that were applicable in the design of antiviral face masks/filters, that is, the 0, 2, and 5% (w/w) of ZnO-NPs, were loaded onto the PVDF solution.

Morphology Investigation of ZnO-Loaded PVDF NFs

The microstructures of both blank PVDF nanofibers and ZnO-loaded PVDF composite nanofibers were investigated from the SEM images displayed in Figure . The obtained nanofiber structure was imbedded with ZnO-NPs that play an important role in antiviral activities, as will be described in Section . An optimum beads-free nanofibrous structure was shown in the case of pure PVDF NFs, while through the addition of ZnO-NPs, the polymeric chain arrangement and crystallography structure might change; these change the electrospinning conditions, resulting in an increase in the encapsulation ratio with an increase in the concentration of ZnO-NPs and decrease in the nanofiber diameter (average of 30 measurements) for PVDF, 2% ZnO/PVDF, and 5% ZnO/PVDF of 194, 164, and 83 nm, respectively. The mixing of ZnO-NPs with the PVDF solution before using the electrospun technique allows the ZnO-NPs to be imbedded and stabilized with the nanofiber interior structure. As ZnO is a semiconductor particle that decreases the dielectric constant of the polymeric solution, it results in the instability of the whip and charge density in the Taylor cone, leading to an increase in the nanofiber diameter and formation of more encapsulated structures under the same condition of electrospinning of pure PVDF. It is noticeable that ZnO-NPs enable the reduction of the critical voltage for electrospinning from 23 to 18 kV.[47] Also, it has to be stated that increasing the solution conductivity to carry more charge results in the formation of thinner nanofibers, and the fibrous diameter reported to be decreased due to the enhanced conductivity of the ZnO/PVDF composite and formation of thinner nanofibers. Therefore the encapsulation of ZnO-NPs has appeared obviously with increasing its content with respect to the fixed percentage of PVDF. The content of ZnO was evaluated through EDX analysis, and the PVDF content evaluated with respect to the wt % of F element is almost stable (65 ± 3 wt %, F). Using PVDF as the carrier for ZnO-NPs, the presence of ZnO in the nanofiber structure also indicates the loading of Zn element in 2% ZnO/PVDF and 5% ZnO/PVDF NFs by 1.14 wt % Zn and 2.2 wt % Zn, respectively. The increasing ZnO content might be implemented on the tubular structure of the nanofibers as well as its outer surface; hence, the antiviral effect is shown with respect to the used loading percentages.

Figure 1

SEM images and EDX analysis of (a) blank PVDF, (b) 2 wt % ZnO/PVDF, (c) 5 wt % ZnO/PVDF composite nanofibers, with the original magnification at 30,000×@10 kV.

Chemical Structure Verification of ZnO-Loaded PVDF NFs

The XRD patterns of ZnO-NPs and the normalized chart of PVDF NFs, 2% ZnO/PVDF NFs, and 5% ZnO/PVDF NFs are presented in Figure a. The standards card number of ZnO NPs is 01-078-2585; the patterns at 2θ = 18.5° and 20.2° were attributed to PVDF crystallites, and the α phase is predominantly PVDF crystalline, with three most distinctive diffraction patterns at 2θ = 18.5°, 20.2°, and 26.6°.[48] Furthermore, the broad pattern in the case of PVDF is due to the formation of a layered structure after the electrospinning process. This layered structure led to the shift of the pattern positions in a broad form;[49] these pattern positions were detected at 2θ = 31.6°, 34.3 °, 36.1°, 47.5°, 56.5°, 62.7°, 66.4°, 67.8°, and 68.9°, which were attributed to the ZnO crystal phases of (100), (002), (101), (102), (110), (103), (200), (112), and (201), respectively.[50] This speculation could confirm the preservation of ZnO-NPs and their chemical composition.

Figure 2

(a) XRD patterns of pure PVDF and ZnO NP-loaded PVDF NFs, (b) FTIR spectra showing no change in the chemical groups after loading ZnO NPs, indicating no chemical interactions.

Figure b shows the FTIR spectra of ZnO-NPs and ZnO-loaded PVDF composite nanofibers. The asymmetric and symmetric vibrations of the C–H bond of PVDF appeared at ν 3022 and 2978 cm–1, respectively. The absorption peaks at ν 1398 and 1176 cm–1 were attributed to the C–F and CF2 stretching peaks, respectively. These peaks at ν 877 and 837 cm–1 were attributed to C–H bending.[51,52] Hence, the antiviral activity could be measured for the ZnO and PVDF nanofiber materials; the elucidation of no chemical interaction between ZnO-NPs and PVDF and also no change in the phase structure of PVDF ensures the antiviral effect which was attributed to ZnO-NPs.

Mechanical Measurements of ZnO-Loaded PVDF NFs

The elongation-at-break (%), Young’s modulus, tensile strength, and elastic modulus of three nanofiber-based mats were measured to evaluate their mechanical stability, as presented in Table . The loading of ZnO-NPs into PVDF NFs noticeably increased their mechanical properties, in particular by incorporating 5 wt % of ZnO-NPs. As the mechanical properties represent an important factor in textile technology, the elongation-at-break (%) of 5% ZnO/PVDF NFs successfully increased to 30 ± 2.5% compared to that of the blank PVDF NFs, which is 24 ± 0.5%. Moreover, Young’s modulus of composite NFs was enhanced by loading 5 wt % ZnO. This was attributed to the presence of ZnO-NPs besides their role as antiviral agents, as will be presented in Section . Accordingly, ZnO-NPs enhance the mechanical properties of the proposed antiviral nanofibers compared to the blank PVDF nanofibers.

Table 1

Mechanical Measurements of Electrospun PVDF and 2% ZnO/PVDF NFs and 5% ZnO/PVDF NFs

test	PVDF NFs	2% ZnO/PVDF NFs	5% ZnO/PVDF NFs
max. strain	24 ± 0.5	25 ± 0.5	30 ± 2.5
max. displacement (mm)	4.8	4.8	6.1
Young’s modulus (MPa)	2501 ± 1.57	1998 ± 2.35	2633 ± 6.5
tensile strength (MPa)	55	56	64
elastic modulus (GPa)	1.5 ± 0.4	1.65 ± 0.52	1.95 ± 0.65

Stability Measurements of ZnO-NPs Loaded PVDF NFs

The stability measurements for color, flexibility, shelf-life after 5 months, and humidity uptake of the fabricated 5 wt % ZnO-loaded PVDF NFs were conducted using a humidity chamber, and the obtained data are listed and shown in Table .

Table 2

Stability Tests of 5% ZnO/PVDF NFs

test	nanofibers after 0 day of fabrication	nanofibers after 5 months of fabrication
nanofiber color	white	white
nanofiber flexibility	flexible	flexible
shelf stability	stable at room temp.	stable at room temp.
humidity uptake	0%	0%a

Humidity uptake after 72 h in a closed system contains 200 mL of H2O.

As shown in Figure S2 (Supporting Information), 5 wt % ZnO-NPs loaded PVDF nanofibers show quite stable appearance and properties even after almost 1 year; also, no humidity absorption was observed due to the high hydrophobicity of PVDF, while the incorporation of ZnO-NPs into PVDF does not have an effect on the wettability of fabricated composite nanofibers. These findings support the use of fabricated composite nanofibers as potential antiviral filters/face masks without the need for special storage conditions.

Zeta-Potential of ZnO-NPs

The measured charge of ZnO-NPs is +11.8 mV; this positive charge of ZnO-NPs offers higher mobility and affinity toward the negative charges of the cell membrane biomolecules, thus interrupting the binding of the virus to host receptors and preventing viral attachment.[53] Moreover, it has been reported that the positive charges of Zn can impair the replication of many RNA viruses, and its incubation with DNA viruses like herpes virus neutralizes its effect and hence prevents its infection.[54]

Cytotoxicity Test of ZnO-Loaded PVDF NFs

Good biocompatibility of PVDF and ZnO-NPs has been stated.[55,56] The cytotoxicity test of fabricated PVDF, 2% ZnO/PVDF NFs, and 5% ZnO/PVDF composite NFs was performed using MTT assay on Vero cells, as shown in Figure . The cell viability (%) values reveal that all fabricated nanofibers and composite nanofibers show safe and nontoxic behavior on Vero cells ranging between 70 and 80%, which can be evaluated for their antiviral activity against ADV5. Meanwhile, the loading of ZnO-NPs into PVDF NFs does not exhibit a noticeable impact on the cell viability behavior, compared to the blank PVDF NF group, Figure .

Figure 3

Cell viability (%) by MTT assay of PVDF, 2% ZnO/PVDF NFs, and 5% ZnO/PVDF NFs.

Antiviral Activity of ZnO-NPs Loaded PVDF NFs against ADV5

It has been reported previously that ZnO-NPs exert their antiviral activity against human influenza virus after the viral entry.[57] Results showed that IC50 was identified at 105 copies/mL of viral load and used in the antiviral assay. The results of antiviral activity in the adsorption mechanism showed that 5% ZnO/PVDF NFs possess antiviral activity, easily preventing viral entry into cells. Moreover, ZnO/PVDF composite nanofibers possess antiviral activity in virucidal mechanism, thus preventing its replication into the cells, as evidenced by the undetected levels using RT-qPCR assay.

Biomedical applications of nanoparticles demonstrate several advantages owing to their size, surface charges, drug loading, antiviral effect, and so forth. Current results show that ADV-5 was titrated using MTT assay to determine the IC50 dose (dilution of virus that can kill 50% of cells). A result showed that virus at dilution of 1:100 is IC50 and was used in the antiviral assay. The results showed that 5% ZnO/PVDF NFs prevent viral entry into host cells (Table .

Table 3

RT-qPCR Assay of ADV-5-Treated Vero Cells with Our Tested Materials

ADV-5	CTa	copies/mLa	CTb	copies/mLb
untreated Vero cells (cell control)	under detection (UD)	UD	UD	UD
PVDF NFs	14.2	7.4 × 107	25.5	1.3 × 103
2% ZnO/PVDF NFs	15.9	5.9 × 106	UD	UD
5% ZnO/PVDF NFs	UD	UD	UD	UD
VC	24.4	3.3 × 103	24.4	3.3 × 103
(+ve control)	22.8	2.7 × 104	22.8	2.7 × 104
(−ve control)	UD	UD	UD	UD

RT-qPCR assay of ADV-5-treated cells (adsorption).

For the against (virucidal) with the selected nontoxic materials. CT, mean threshold cycle; VC, virus control.

The proposed mechanisms for explaining the antiviral activity of ZnO-NPs could be stated as follows:

(i) The ability of nanostructures to neutralize the virus and the physical entrapment exerted by zinc nanoparticles besides the electrostatic interference of H (−OH group-rich) zinc nanoparticles are more significant to decrease the toxicity and increase the antiviral activity.[54] (ii) The toxicological effects of ZnO-NPs are due to the Zn2+ ions released from the aqueous dissolution of ZnO-NPs (ZnO + H2O ⇄ Zn2+ + 2OH–). However, such mechanism still remains unclear. Interestingly, it is suggested that the antiviral effect of ZnO-NPs is due to the oxidative stress induced in response to Zn oxide causing a damage to viral DNA, thus preventing viral replication.[58] (iii) Zn2+ ions inhibit the viral entry, local replication, and spread to cells during the viral pathogenesis process, which has been previously described for the antiviral activity against HSV. Also, the nanostructure affinities toward different surface viral receptors might prevent the viral entry into the cells.[54,58] Another proposed mechanism is the transient elevation of Zn2+ concentration that facilitates its complexation with the adhesion molecules on the cell surface, preventing the binding of the virus and its entry, as previously proposed for the antiviral activity against rhinoviruses.[59] (iv) ZnO micro–nanostructures exert an inhibitory effect on HSV-1 due to their direct interaction with the viral particles, trapping the virions, and subsequently, blocking the HSV entry into their target cells. A similar mechanism can be suggested for our used model ADV-5 via the virucidal mechanism, after incubating the virus with our materials for 1 h before infecting the Vero cells.[60] (v) Another study also showed that ZnO-NPs significantly increased the transcriptional levels of peroxidases (POD) that increase the plant defense mechanism against Tobacco mosaic viruses, providing another possible explanation.[61]

Molecular Docking Calculation of the ZnO@PVDF Polymer against the Hexon Protein in ADV-5

Molecular docking simulations revealed that ligands have a favorable interaction with the hexon protein (PDB ID: 6CGV). In the estimated binding pocket, the PVDF molecule had the highest binding affinity (−7.1 kcal/mol) against the hexon protein. However, the ZnO@PVDF molecule binds to ACE2 with similar energies of around −6.7 kcal/mol. The protein–ligand interaction profiler (PLIP) analysis online tool was used to look at the interactions of the two ligands in the estimated binding pockets (Figures and 5). Hydrophobic interactions dominated the PVDF interactions with the hexon protein, and it is calculated deeply in the Supporting Information. Between LYS44B in the hexon protein and the Fluor atom in PVDF, one halogen bond was formed in the estimated binding pose (the pose with the maximum binding affinity). Furthermore, the hydrophobic interaction of ZnO@PVDF with the hexon protein was found to be the most important. Between the Fluor atom and LEU 28B, one halogen bond was created. Furthermore, in the hexon protein, ZnO forms two hydrogen bonds with 44B LYS and 45B PHE.

Figure 4

Interaction of PVDF with the hexon protein in the predicted binding pocket.

Figure 5

Interaction of ZnO@PVDF with the hexon protein in the predicted binding pocket.

Conclusions

Antiviral face masks/filters that demolish adenovirus and prevent the spreading of respiratory viruses that have been targeted via the loading of 0, 2, and 5% (w/w) ZnO-NPs onto electrospun PVDF nanofibers have been successfully designed. The one-layered structure of fabricated ZnO-NPs@ PVDF NFs is cost-effective through the reduction of the applied voltage from 23 to 18 kV; also, the mechanical properties of the produced nanofibers are enhanced with an increase in the incorporated ZnO content. In applying two antiviral mechanisms, the pure PVDF NFs showed no antiviral effect against adenovirus in both the virucidal and adsorption mechanisms; however, the loading with 2% ZnO-NPs@PVDF NFs showed effective antiviral behavior only with the virucidal mechanism, while the loading with 5% ZnO-NPs@PVDF NFs exhibited a significant and high antiviral effect against both applied mechanisms. Accordingly, ZnO-NPs loaded electrospun PVDF NFs are highly recommended for use as efficient antiviral filters for face masks that work on the suppression of respiratory infectious diseases.