Antimicrobial Nanofiber Based Filters for High Filtration Efficiency

Article Antimicrobial Nanoﬁber Based Filters for High Filtration Efﬁciency Respirators

Maria Pardo-Figuerez 1,2 , Alberto Chiva-Flor 2, Kelly Figueroa-Lopez 1 , Cristina Prieto 1 and Jose M. Lagaron 1,*

1 Novel Materials and Nanotechnology Group, Institute of Agrochemistry and Food Technology (IATA), Spanish Council for Scientiﬁc Research (CSIC), Calle Catedrático Agustín Escardino Benlloch 7, 46980 Paterna, Spain; [email protected] (M.P.-F.); kjﬁ[email protected] (K.F.-L.); [email protected] (C.P.) 2 Bioinicia S.L., R & D Department, Calle Algepser, 65 Nave 3, 46980 Paterna, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-963-900-022

Abstract: Electrospinning has been used to develop and upscale polyacrylonitrile (PAN) nanofibers as effective aerosol filtration materials for their potential use in respirators. The fibers were deposited onto non-woven spunbond polypropylene (SPP) and the basis weight (grammage, g/m2) was varied to assess the resulting effect on filtration efficiency and breathing resistance of the materials. The results indicated that a basis weight in excess of 0.4 g/m2 of PAN electrospun fibers yielded a filtration efficiency over 97%, with breathing resistance values that increased proportionally with the amount of basis weight added. With the aim of retaining filter efficiency whilst lowering breathing resistance, the basis weight of 0.4 g/m2 and 0.8 g/m2 of PAN electrospun fibers were strategically split up and stacked with SPP in different configurations. The results suggested that a symmetric structure based on SPP/PAN/PAN/SPP was the optimal structure, as it reduces SPP consumption Citation: Pardo-Figuerez, M.; Chiva-Flor, A.; Figueroa-Lopez, K.; while maintaining an FFP2-type of filtration efficiency, while reducing breathing resistance, specially Prieto, C.; Lagaron, J.M. at high air flow rates, such as those mimicking FFP2 exhalation conditions. The incorporation of zinc Antimicrobial Nanofiber Based Filters oxide (ZnO) nanoparticles within the electrospun nanofibers in the form of nanocomposites, retained for High Filtration Efficiency the high filtration characteristics of the unfilled filter, while exhibiting a strong bactericidal capacity, Respirators. Nanomaterials 2021, 11, even after short contact times. This study demonstrates the potential of using the symmetric splitting 900. https://doi.org/10.3390/nano of the PAN nanofibers layer as a somewhat more efficient configuration in the design of filters for 11040900 respirators.

Academic Editor: Takuya Kitaoka Keywords: nanoﬁbers; respirators; antimicrobials; SARS-CoV-2; electrospinning

Received: 21 February 2021 Accepted: 29 March 2021 Published: 1 April 2021 1. Introduction

Publisher’s Note: MDPI stays neutral The unexpected appearance and rapid spreading of the severe acute respiratory with regard to jurisdictional claims in syndrome coronavirus (SARS-CoV-2) at the end of 2019, has affected over 14 million people published maps and institutional affil- and caused over a million deaths all over the world as of November 2020 [1–3]. The SARS- iations. CoV-2 belongs to the coronavirus family, a type of positive sense single-stranded RNA virus with a spike of glycoproteins on its envelope which gives the virus a crown-like morphology, with a size ranging from 60 to 140 nm [4,5]. The first reported cases indicated that SARS-CoV-2 affects mainly the respiratory tracks, with patients reporting mostly fever, cough, loss of smell and taste, headache and fatigue [6]. The exported cases from Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. the epicenter of the pandemic also suggested that human-to-human transmission occurs This article is an open access article rapidly. It is believed that virus spreading occurs through different means, being the distributed under the terms and inhalation of mouth-expelled microdroplets of over 5 µm and aerosols below 5 µm, as the conditions of the Creative Commons most recently recognized mechanism [1,5]. Due to the fast spreading, authorities all over the Attribution (CC BY) license (https:// globe have focused on reducing the transmission by practicing isolation, social distancing creativecommons.org/licenses/by/ and the use of face masks and respirators [5,7]. Although isolation measures have proven 4.0/). effective, countries cannot be confined for a long term due to socio-economic reasons [8]

Nanomaterials 2021, 11, 900. https://doi.org/10.3390/nano11040900 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, 900 2 of 17

and therefore, authorities have recommended, and even declared compulsory [9], the use of face masks and respirators to avoid the spreading of such global infections whilst avoiding recession and financial collapse. In light of this, there is an urgent need to generate highly efficient filtration materials that can help from a prophylactic view-point to fight the current pandemic [1,5]. Current commercial filters are mainly microfiber-based materials, being the most efficient meltblown polypropylene. Although frequently used, meltblown polypropylene presents uncertain durability while in use, due to moisture-induced dissipation of the electrostatic filtration mechanism in which the technology is based [10–12]. Electrospinning is becoming an emerging technology in the area of filtration to generate nanofiber-based filter materials. These fibrous materials at the nano scale have significant morphological advantages over current conventional filters, such as fiber diameters within the nanoscale, homogeneous and interconnected small pore size, good mechanical properties and a high specific surface area of the fibers. These characteristics favor a mechanical filtration mechanism, where interception and Brownian diffusion retention of small particles between 50–5000 nm result in highly efficient filtering materials [11,13]. Due to these features, the nanofiber technology also provides a high filter efficiency at very low grammages, structural simplicity and the possibility of functionalization of the nanofibers to add new active performances. This makes this technology cost-effective, competitive and more functional when compared to, for instance, electrostatic meltblown microfibers used in conventional filters [11]. Electrospun nanofibers are normally deposited onto a substrate, commonly a non- woven fabric [14]. Once an optimum substrate has been chosen, electrospun fibers can be designed to increase filter efficiency whilst maintaining a low breathing resistance. This can be typically achieved either by modification of interfiber porosity or by increasing the basis weight of the nanofibers layer. The latter may not be an optimal strategy since a larger nanofiber mass deposition will lead to a higher filter efficiency but also to a higher pressure drop, obtaining poor breathability. Various strategies have been presented in the literature to try and achieve efficient filtration nanofiber materials. In a study from Leung and Sun [15], the filtration efficiency of untreated and corona-treated (charged) polypropylene deposited-polyvinylidene fluoride (PVDF) fibers with a size diameter of 450 nm was analyzed. The study confirmed that corona treatment could favor high filtration efficiency due to high charge densities on filters, leading to a stronger electrostatic mechanism. However, filter performance did not increase when the authors attempted to increase the filter basis weight, and so to enhance the filter electrical mechanisms, the filter was divided into multiple layers. The stacking up of the filter not only increased filter efficiency, but also lowered pressure drop. This effect was more notable at higher basis weight, leading to an overall increase on the quality factor of the filter [15]. Similarly, filters of PVDF electrospun fibers were produced with different fiber diameters (84, 191, 349 and 525 nm) and were also corona-discharged to enhance differences between mechanical and electrostatic capture [16]. Filter efficiency and pressure drop were assessed with sodium aerosols against single and multi-nanofiber layers deposited in polypropylene, by stacking each nanofiber with its SPP substrate in several configurations, ranging from 2 to 8 layers. The authors observed that using the multilayer approach reduced pressure drop significantly when compared to the monolayer structures at the same basis weight. It was hypothesized that this could be attributed to the introduction of macropores from the substrate material layer to the filter, which would interrupt the micropore structure of the multilayer nanofibers and thus reduce the fiber packing density. The benefits of the multilayer structure versus the monolayer could be demonstrated for the four different fiber diameters tested, although changes in the basis weight of the modules had to be adjusted for each fiber diameter [16]. Among the different types of polymers used in filtration, polyacrylonitrile (PAN) has become a good candidate due to its good mechanical properties and hydrophobicity, as well as both its thermal and chemical stability [17–19]. A comparison of the filtration Nanomaterials 2021, 11, 900 3 of 17

performance between single and bilayer electrospun filters on a polyester–viscose non- woven substrate was also analyzed for the PAN fibers. In this case, the bilayer structure consisted of a layer of smooth fibers with 400 nm diameter, and another layer containing beaded structures (beads, 200 nm, strings 55 nm). The change in morphology across the layers altered the thickness and the packing density of the filter. Interestingly, the order of the beaded bilayer also influenced filter performance. When the beaded fiber layer was on the upper layer of the filter, filtration efficiency was above 95% and the pressure drop was 137 Pa. When the beaded fiber layer was placed in the inner layer, penetration performance was similar (95%); however, the pressure drop reached 112 Pa [11]. The same group carried out a similar study in which the bilayer structure was made of smooth nanofiber layers of 200 and 400 nm fiber diameters over a polyester-viscose substrate. The bilayer structures proved beneficial over single layer structures and it was observed that the stacking order in the bilayer could influence the filter performance. The bilayer structure with 400 nm fiber diameter at the top and the 200 nm fiber diameter layer at the bottom of the filter showed a significant reduction in pressure drop (from 208 Pa to 87 Pa) but comparable filtration efficiency [20]. PAN electrospun fibers have been used on its own as filtration membranes, but this material has also been conveniently additivated with certain agents to enhance fiber functionality, especially in the field of sensors, filtration, catalysis, or food packaging [21–23]. Components such as silver (Ag) [24,25], titanium dioxide (TiO2)[26], palladium (Pd) [27], zinc oxide (ZnO) [28,29], or copper (Cu) [30] have shown good antimicrobial properties against a large variety of microorganisms such as bacteria, fungi, and viruses [31,32] and can be incorporated in the electrospun material in the form of nanocomposites. In a recent study, Bechelany et al. [23] prepared TiO2/PAN, ZnO/PAN, and Ag/PAN nanofibers over polyethylene terephthalate (PET) substrates for their use as air filters. The presence of nanoparticles did not alter the filtration efficiency, as all of them had a filtration above 95%. Additionally, the Ag/PAN filter was the most optimum filter as it showed a high filtration efficiency (>98%) and a low pressure drop (68.13 Pa), as well as very good antibacterial properties. The combination of electrospun fibers with antimicrobial components present a promising prospect in obtaining a highly efficient filtration materials that are able to prevent microorganism growth and contamination, thus protecting the citizens from microbial pollution [30]. This paper reports work carried out to develop high throughput antimicrobial electrospun filter structures made of PAN nanofibers. To do this, we characterized first the filtration efficiency and breathing resistance as a function of the increasing PAN nanofibers bases weight. Furthermore, we proposed the use of the innovative approach of symmetri- cal splitting up of the PAN electrospun nanofibers to yield an optimum balance between the pressure drop (breathability) and the filtration of the material. To impart biocide performance, the PAN electrospun fibers were loaded with ZnO nanoparticles, exhibiting antimicrobial activity without compromising filtration efficiency. The herein developed filtration membranes could open up new avenues for the development of the next generation of ultrahigh filtration materials. Finally, the development and manufacturing of these membranes were obtained in a high throughput industrial equipment, which further indicates that mass production of such materials is feasible.

2. Materials and Methods 2.1. Materials Polyacrylonitrile (PAN, Mw 150.000, 99%) was purchased from Carbosynth (Compton, United Kingdom). Zinc Oxide (CR-4FCC1), 99% purity, specific surface of 4.5 m2/g, bulk density of 40 lb/ft3, and specific gravity of 5.6 were obtained from GH Chemicals LTD® (Quebec, Canada) and N, N-Dimethylformamide (DMF, 99%) was purchased from VWR Chemicals (Leuven, Belgium). All the electrospun fibers were deposited over a porous non-woven spunbond polypropylene material of 30 g/m2 (SPP, NVEVOLUTIA, Valencia, Spain). All reagents were used as received without further purification. Nanomaterials 2021, 11, 900 4 of 17

2.2. Solution Preparation and Characterization Polyacrylonitrile was mixed with DMF at a concentration of 11 wt.% and dissolved by stirring at 450 rpm and at 50 ◦C overnight. For the nanocomposites ﬁbers, solutions of PAN with DMF (11 wt.%) containing 1 wt.% (PAN-ZnO1), 3 wt.% (PAN-ZnO3), 10 wt.% (PAN-ZnO10) and 20 wt.% (PAN-ZnO20) of ZnO were stirred overnight and homogenized in a high power ultrasound SONOPULS HD 2200.2. (Scharlab, Barcelona, Spain) at 40% power for 2 min. The apparent viscosity (ηa) was determined using a rotational viscosity meter Visco BasicPlus L from Fungilab S.A. at 50 rpm with a L3 spindle (Barcelona, Spain). The conductivity was evaluated using a conductivity meter Seven2Go™ from Metler Toledo 742-ISM (Barcelona, Spain). All the measurements were carried out in triplicate at room temperature.

2.3. Electrospinning The electrospun PAN ﬁbers mat and its antimicrobial nanocomposites were processed using a Fluidnatek LE-500 production equipment with an industrial 50 cm wide roll-to-roll system. The solutions were pumped through a linear multinozzle injector at a feed rate of 1 mL/h per emitter, applying a voltage of 30 kV at the emitter and a voltage of −10 kV at the collector. The ﬁbers were collected onto the spunbond polypropylene (SPP) at a distance of 20 cm under controlled environmental conditions of 30 ◦C and 30% RH (relative humidity). The conditions for electrospinning PAN-ZnO were the same as mentioned above for the pristine PAN electrospun mats. To obtain the different basis weight of the mats (grammage, g/m2), the deposition time was optimized and adjusted accordingly.

2.4. Mat Morphology The morphology of the electrospun fibers was determined by a field emission scanning electron microscope (FE-SEM) using a Hitachi TM-4000 (Tokyo, Japan) with an electron beam acceleration of 10 kV. Analysis of the fiber diameter was carried out with the ImageJ software (version 1.52, National Institutes of Health, Bethesda, MD, USA). At least 100 fibers were randomly selected and measured from three different images. The average fiber diameter as well as the standard deviation (SD) were calculated and are presented in the corresponding Figures. The dispersion of ZnO in the matrix of the fibers was analyzed by transmission electron microscopy (TEM) using a JEOL JEM 1010 (JEOL Ltd., Tokyo, Japan) with an acceleration voltage of 100 kV. The fibers were collected on a sandwich-type holder (Agar Scientific-G230, Agar Scientific Ltd., Stansted, UK) with a mesh size of 3.05 mm. Porosity of the fiber mats was determined by cutting out sample sizes of 3 × 3 cm2, obtaining the mass with a Mettler Toledo MS105 balance and the thickness by measur- ing the cross-section of the samples with a field emission scanning electron microscope (FE-SEM) using a Hitachi TM-4000 (Tokyo, Japan). The apparent density was then measured with the collected data (see Equation (1)) and the porosity was determined using the bulk density of PAN (1.184 g/cm3, see Equation (2)):

weight of the mat Apparent density = (1) thickness ∗ area of the mat

Apparent density Porosity (%) = 1 − × 100 (2) Bulk density Nanomaterials 2021, 11, 900 5 of 17

2.5. Antimicrobial Activity Antimicrobial performance was analyzed for the composite electrospun ﬁlters. The strains Staphylococcus aureus CECT240 (ATCC 6538p) and Escherichia coli CECT434 (ATCC 25922) were obtained from the Spanish Type Culture Collection (CECT, Valencia, Spain). The bacterial strains were stored in phosphate-buffered saline (PBS) with 10 wt.% tryptic soy broth (TSB) obtained from Conda Laboratories (Madrid, Spain) and 10 wt.% glycerol at −80 ◦C. Previous to each study, a loopful of bacteria was transferred to 10 mL of TSB and incubated at 37 ◦C for 24 h. A 100 µL aliquot from the culture was again transferred to TSB and grown at 37 ◦C to the mid-exponential phase of growth. The optical density showing an absorbance value of 0.20 and measured at 600 nm in a UV–Vis spectrophotometer (VIS3000 Dinko Instruments, Barcelona, Spain) determined that the initial bacterial concentration was approximately a 5 × 105 colony-forming unit (CFU)/mL. The antimicrobial performance of PAN ﬁlters containing ZnO particles (1, 3, 10 and 20 wt.%) was determined based on the guidelines of the Japanese Industrial Standard JIS Z2801 (ISO 22196:2007) [33]. A microorganism suspension in TSB of S. aureus and E. coli was applied to a 5 cm × 5 cm material sample containing PAN only (negative control), and PAN-loaded ZnO particles at 1, 3, 10, and 20 wt.% (test material). Thereafter, the inoculated samples were placed in open bottles and incubated for 24 h at 24 ◦C and at a relative humidity (R.H.) of at least 95%. For the PAN containing a 3 wt.% of ZnO, samples were also incubated for 1, 3, 6, and 8 h. Bacteria were recovered with PBS, 10-fold serially diluted and incubated for 24 h at 37 ◦C to quantify the number of viable bacteria by a conventional plate count. The antimicrobial activity reduction (R) was evaluated at 24 h using Equation (3):

B C B R = Log − Log = Log (3) A A C

where A is the average of the number of viable bacteria on the control sample immediately after inoculation, B is the average of the number of viable bacteria on the control sample after 24 h, and C is the average of the number of viable bacteria on the test sample after 24 h. The antibacterial activity of the filters was assessed as a non-significant reduction if R < 0.5, a slight reduction if R ≥ 0.5 and <1, a significant reduction when R ≥ 1 and <3, and a strong reduction if R ≥ 3. Experiments were performed in triplicate [34].

2.6. Filter Performance The filtration and breathing resistance performance of the filter was measured using the procedures gathered within the European EN149:2001+A1:2009 standard using a Filter Media Test Rig PMFT 1000 M (Palas®, Karlsruhe, Germany). The penetration test was carried out with a paraffin oil aerosol with particle sizes spanning from ca. 0.140 to 4 µm, after 3 min of aerosol exposure. The pressure drop of the samples, named in this work as breathing resistance (Pa), was measured by passing compressed air through the filter at flow rates of 30 and 95 L/min simulating inhalation, and at 160 L/min simulating exhalation in the opposite direction. The dimension of the samples measured was approximately 50 cm2 and the testing conditions were 24 ± 2 ◦C and 50 ± 10% relative humidity. According to the abovementioned EN149 standard, final respirators should have a filter efficiency of 94% or higher to the paraffin aerosol and a pressure drop limit of 70 Pa at 30 L/min, of 240 Pa at 95 L/min during inhalation, and of 300 Pa at 160 L/min during exhalation, to accredit FFP2 like performance. FFP2 or its equivalent is the most recom- mendable filter according to the Health Authorities to protect, especially for healthcare professionals, from COVID-19 [35]. Since pressure drop has a linear relationship with the sample area and most commercial respirators have respiration areas bigger than 200 cm2, the breathing resistance data measured in the 50 cm2 sample areas should be effectively divided by a factor of at least 4 to assess their compliance with FFP2 performance. Nanomaterials 2021, 11, x FOR PEER REVIEW 6 of 17

3. Results and Discussion NanomaterialsNanomaterials 20212021,, 1111,, x 900 FOR PEER REVIEW 66 of of 17 17 3.1. Fiber Morphology Figure 1 shows the morphology of the generated PAN fibers by FE-SEM. Initially, electrospun3.3. Results Results and and mats Discussion Discussion were deposited onto non-porous substrates until electrospinning pa- rameters3.1.3.1. Fiber Fiber Morphologywere Morphology optimized. The images show randomly oriented, ultrafine and smooth fiber morphologiesFigureFigure 1 shows with the morphologyan average diameter of the generated within PANthe range fibers fibers of by 200–300 FE-SEM. nm. Initially, electrospunelectrospun mats were depositeddeposited ontoonto non-porous non-porous substrates substrates until unt electrospinningil electrospinning param- pa- rameterseters were were optimized. optimized. The The images images show show randomly randomly oriented, oriented, ultrafine ultrafine and and smooth smooth fiber fi- bermorphologies morphologies with with an averagean average diameter diameter within within the the range range of 200–300 of 200–300 nm. nm.

Figure 1. (A) Field emission scanning electron microscope (FE-SEM) micrographs of polyacrylonitrile (PAN) electrospun fibers at 3000×. (B) Field emission scanning electron microscope (FE-SEM) micrographsFigureFigure 1. 1. ((AA)) Field Fieldof polyacrylonitrile emission emission scanning scanning (PAN) electron electrospun microscopemicroscope fibers (FE-SEM) (FE-SEM) at electrospun micrographs micrographs fibers of of polyacrylonitrile polyacryloni- at 8000×. trile(PAN) (PAN) electrospun electrospun fibers fibers at 3000at 3000×.×.( B(B)) FieldField emission scanning scanning electron electron microscope microscope (FE-SEM) (FE-SEM) micrographs of polyacrylonitrile (PAN) electrospun fibers at electrospun fibers at 8000×.× micrographsThe optimized of polyacrylonitrile electrospinning (PAN) electrospun parameters fibers were at electrospun utilized fibers to deposit at 8000 PAN. nanofibers onto ThetheThe optimizednon-woven optimized electrospinning electrospinning polypropylene parameters parameters substrate were were (SPP). utilized utilized Figure to to deposit deposit 2A shows PAN PAN nanofibersthe nanofibers morphology, bothontoonto atthe the the non-woven non-woven macroscopical polypropylene polypropylene and microscopical substrate substrate (SPP). (SPP).level, Figure of Figure the electrospun2A2A shows the fibers morphology, deposited onto thebothboth SPP. at at the the Smooth macroscopical macroscopical nanometric and and microscopical microscopical fibers were le level,obtavel, ofined of the the electrospunon electrospun top of the fibers fibers SPP deposited deposited microfibers, onto onto which canthethe SPP.be SPP. discerned Smooth Smooth nanometric nanometricdue to the fibers fiberslow basis were were weigobta obtainedinedht deposition on on top top of of the theof theSPP SPP nanofibers microfibers, microfibers, (0.4 which which g/m 2). Fig- 2 urecancan 2Bbe be discernedrepresents discerned due duethe to fiber to the the low size low basis distribution basis weig weightht deposition of deposition the electrospun of the of thenanofibers nanofibers fibers (0.4 onto (0.4g/m the2 g/m). non-wovenFig-). substrate,ureFigure 2B 2representsB representsindicating the the fiber an fiber average size size distribution distribution size of 282.5 of of the the ± electrospun47.6 electrospun nm. The fibe fibers users onto ontoof nanometric the the non-woven fibers have substrate, indicating an average size of 282.5 ± 47.6 nm. The use of nanometric fibers provensubstrate, advantageous indicating an averagein air filtration, size of 282.5 since ± 47.6 their nm. small The use diameter of nanometric and high fibers surface have to vol- have proven advantageous in air filtration, since their small diameter and high surface to umeproven ratios, advantageous can enhance in air the filtration, capture since of particle their smalls through diameter interception and high surface [13,16]. to vol-As well as umevolume ratios, ratios, can can enhance enhance the the capture capture of ofparticle particless through through interception interception [13,16]. [13,16]. As As well well as as this, the homogeneous porosity and slip flow effect of the nanofibers will certainly result this,this, the the homogeneous homogeneous porosity andand slipslip flowflow effect effect of of the the nanofibers nanofibers will will certainly certainly result result in in a lower pressure drop than their microfibers counterparts [36]. These characteristics ina lowera lower pressure pressure drop drop than than their their microfibers microfib counterpartsers counterparts [36]. [36]. These These characteristics characteristics make makemakeelectrospun electrospun nanofibers nanofibers nanofibers very attractivevery very attractive attractive in the in filtration the in filtrationthe field. filtration field. field.

FigureFigure 2. 2. (A(A) )Macroscopic Macroscopic and and microscopic microscopic images images of of the the spunbound spunbound polypropylene polypropylene (SPP) (SPP) coated coated Figurewith the 2. with (A) Macroscopicpolyacrylonitrile and (PAN) microscopic electrospun images fibers. of ( Bthe) Average spunbound fiber distribution polypropylene of polyac- (SPP) coated with the with polyacrylonitrile (PAN) electrospun fibers. (B) Average fiber distribution of polyacry- withrylonitrile the with (PAN) polyacrylonitrile electrospun fibers. (PAN) electrospun fibers. (B) Average fiber distribution of polyac- lonitrile (PAN) electrospun fibers. rylonitrile (PAN) electrospun fibers.

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Nanomaterials 2021, 11, 900 7 of 17

3.2. Filter Performance Using Mono and Multilayer Electrospun Fibers It is known that when the basis weight is increased in filters, dramatic changes in 3.2. Filter Performance Using Mono and Multilayer Electrospun Fibers filter efficiency may occur, especially for nanofibrous filters, where a low basis weight is required forIt is high known filtration that when performance the basis and weight low pressure is increased drop in [16,20,37,38]. filters, dramatic In fact, changes the in use offilter nanofibers efficiency in mayair filtration occur, especially applications for nanofibrous requires much filters, less where basis aweight low basis than weight the is microfibrous-basedrequired for high filters filtration [11,16,39] performance and hence, and the low optimum pressure basis drop weight [16,20 ,of37 PAN,38]. Infibers fact, the to obtainuse ofa high nanofibers filter efficiency in air filtration and low applications breathing resistance requires was much investigated less basis (Figure weight 3A). than the microfibrous-based filters [11,16,39] and hence, the optimum basis weight of PAN fibers to To do this, a mass deposition of nanofibers ranging from 0.1–1 g/m2 were deposited on an obtain a high filter efficiency and low breathing resistance was investigated (Figure3A). SPP by electrospinning. Thereafter, another SPP was placed over the electrospun deposi- To do this, a mass deposition of nanofibers ranging from 0.1–1 g/m2 were deposited on an tion to protect the PAN fibers and to provide better structural rigidity to the filter (Figure SPP by electrospinning. Thereafter, another SPP was placed over the electrospun deposition 3B) [17]. Figure 3A shows the relationship between the basis weight against the filter effi- to protect the PAN fibers and to provide better structural rigidity to the filter (Figure3B) [ 17]. ciency and breathing resistance. Initially, two layers of SPP/SPP without nanofibers were Figure3A shows the relationship between the basis weight against the filter efficiency and measured as the reference system. The filter efficiency was very poor, with a value of ap- breathing resistance. Initially, two layers of SPP/SPP without nanofibers were measured as proximately 2.62 ± 1.78%. In terms of the breathing resistance, the value was below 20 Pa the reference system. The filter efficiency was very poor, with a value of approximately for all the flow rates tested. Once the PAN electrospun fibers were added to the substrate, 2.62 ± 1.78%. In terms of the breathing resistance, the value was below 20 Pa for all the an increase in filtration efficiency of up to 60–70% was observed by using only 0.1 g/m2. flow rates tested. Once the PAN electrospun fibers were added to the substrate, an increase As the basis weight increased, so did the filter efficiency, reaching a 97% at 0.4 g/m2 and in filtration efficiency of up to 60–70% was observed by using only 0.1 g/m2. As the basis 2 maintainingweight increased,between 97–99% so did thefor filterthe 0.8 efficiency, and 1 g/m reaching basis weight. a 97% at As 0.4 expected, g/m2 and the maintaining higher the filtrationbetween efficiency, 97–99% for the the hi 0.8gher and the 1 g/mbreathing2 basis resistance weight. As achieved expected, for thethe higherthree different the filtration 2 air flowsefficiency, (30, 95 the and higher 160 L/min), the breathing with values resistance reaching achieved their for maximum the three at different 1 g/m basis air flows 2 weight.(30, It 95 should and 160 be L/min),borne in withmind values that testing reaching was their done maximum in filter areas at 1 of g/m 50 2cmbasis; thus, weight. 2 althoughIt should filtration be borne efficiency in mind should that not, testing and was has donenot been in filter seen areas (i.e., 50 of vs. 50 cm1002 ;cm thus,, results although not shown),filtration to efficiency depend on should filter area, not, andbreathing has not resistance been seen is expected (i.e., 50 vs.to decrease 100 cm2 ,linearly results not withshown), the increasing to depend sample on filter area. area, Respirators breathing for resistance personal isprotection expected typically to decrease have linearly areas with largerthe than increasing 200 cm2 sample, so to anticipate area. Respirators the actual for breath personaling protectionresistance typicallyon a typical have final areas res- larger pirator,than these 200 cmvalues2, so should to anticipate be divi theded actual by, at breathingleast, a factor resistance of 4. on a typical final respirator, these values should be divided by, at least, a factor of 4.

Figure 3. (A) Filtration efficiency and breathing resistance of control (SPP/SPP) and monolayer Figurespunbond 3. (A) Filtration polypropylene/polyacrylonitrile/spunbond efficiency and breathing resistance of polypropylenecontrol (SPP/SPP) (SPP/PAN/SPP) and monolayer filter with spunbondvarious polypropylene/polyacrylonitrile/spunb nanofiber basis weights (g/m2). Resultsond are polypropylene presented as the (SPP/PAN/SPP) mean ± SD (n filter= 3). with (B) Diagram of spunbond polypropylene/polyacrylonitrile/spunbond polypropylene (SPP/PAN/SPP) filters with electrospun nanofibers.

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various nanofiber basis weights (g/m2). Results are presented as the mean ± SD (n = 3). (B) Dia- gram of spunbond polypropylene/polyacrylonitrile/spunbond polypropylene (SPP/PAN/SPP) filters with electrospun nanofibers.

Nanomaterials 2021, 11, 900 Filter efficiency and, therefore, breathing resistance, can be tailored by either8 of 17 modi- fying the fiber diameter, packing density and/or the fiber morphology of the electrospun mat. In this case, increasing the deposited mass of nanofibers with the same fiber diameter increasedFilter filter efficiency efficiency, and, but therefore, also led breathing to anresistance, increase canin the betailored packing by density, either modify- which ulti- matelying increased the fiber diameter, breathing packing resistance. density Althou and/or thegh most fiber morphology of the approaches of the electrospun for electrospun filtersmat. are In based this case, on increasing single layers the deposited of nanofibers, mass of nanofibersthe arrangement with the same of the fiber nanofiber diameter layers increased filter efficiency, but also led to an increase in the packing density, which ulti- havemately shown increased certain breathing improvements resistance. in Althoughfiltration most and ofpressure the approaches drop [1,11,20,40]. for electrospun Thus, in orderfilters to retain are based filtration on single efficiency layers of nanofibers,and reduce the breathing arrangement resistance, of the nanofiber a combination layers have of nan- ofibershown layers, certain in alternate improvements and symmetric in filtration stacking and pressure configurations, drop [1,11,20, 40was]. Thus, studied. in order Thus, layers ofto PAN retain filtrationelectrospun efficiency fibers and were reduce strategi breathingcally resistance, disposed a combinationin a multilayer of nanofiber sandwich approach,layers, as indepicted alternate in and Figure symmetric 4. For stacking the mu configurations,ltilayer stacked was studied.structures, Thus, the layers use of of PAN PAN electrospun fibers were strategically disposed in a multilayer sandwich approach, as electrospun fibers with a basis weight of 0.4 and 0.8 g/m2 were used, as these resulted in depicted in Figure4. For the multilayer stacked structures, the use of PAN electrospun highfibers filtration with amaterials basis weight as of monolayers, 0.4 and 0.8 g/m and2 were a pressure used, as these drop resulted was deemed in high filtration acceptable if extrapolatedmaterials asto monolayers,200 cm2 areas and (i.e., a pressure area of drop a typical was deemed respirator). acceptable if extrapolated to 200 cm2 areas (i.e., area of a typical respirator).

Figure 4. Schematic of the mono and multilayer structures of spunbond polypropy- Figure 4. Schematic of the mono and multilayer structures of spunbond polypropylene/polyacry- lene/polyacrylonitrile/spunbond polypropylene (SPP/PAN/SPP), spunbond polypropy- lonitrile/spunbondlene/polyacrylonitrile/polyacrylonitrile/spunbond polypropylene (SPP/PAN/SPP), spunbond polypropylene polypropylene/polyacryloni- (SPP/PAN/PAN/SPP) and trile/polyacrylonitrile/spunbondspunbond polypropylene/polyacrylonitrile/spunbond polypropylene (SPP/PAN/PAN/SPP) polypropylene/polyacrylonitrile/spunbond and spunbond polypropylene/polyacrylonitrile/spunbondpolypropylene (SPP/PAN/SPP/PAN/SPP) polypropylen filters,e/polyacrylonitrile/spunbond prepared with PAN electrospun polypropylene nanofibers. (SPP/PAN/SPP/PAN/SPP) filters, prepared with PAN electrospun nanofibers. The results in filtration efficiency of the multilayer structures can be seen in 2 TableThe 1results. For mono in filtration and multilayer efficiency samples of the prepared multilayer with structures a total of 0.8 can g/m be seenof basis in Table 1. weight (distributed as 0.8 g/m2 for the monolayer and 0.4/0.4 g/m2 for the multilayer For mono and multilayer samples prepared with a total of 0.8 g/m2 of basis weight (dis- filters), a filtration efficiency over 99% was achieved, regardless of the layer distribution. 2 2 tributedFor the as distribution0.8 g/m for of the 0.2/0.2 monolayer g/m2, the and values 0.4/0.4 were g/m between for 97–98%,the multilayer with very filters), similar a filtration valuesefficiency across over the different99% was stacked achieved, structures. regardless of the layer distribution. For the distribution of 0.2/0.2 g/m2, the values were between 97–98%, with very similar values across Table 1. Filtration efficiency (%) of the mono and multilayer configurations. Results are presented as the different stacked structures. the mean ± SD (n = 3).

Table 1. FiltrationSample efficiency (%) of the Basismono Weight and mu (g/mltilayer2) configurations.Filtration Efficiency Results (%)are presented as the mean SPP/PAN/SPP± SD (n = 3). 0.8 99.84 ± 0.02 SPP/PAN/PAN/SPP 0.4/0.4 99.86 ± 0.03 SPP/PAN/SPP/PAN/SPPSample Basis 0.4/0.4Weight (g/m2) Filtration 99.91 ± 0.04 Efficiency (%) SPP/PAN/SPPSPP/PAN/SPP 0.4 0.8 97.95 ± 99.840.46 ± 0.02 SPP/PAN/PAN/SPP 0.2/0.2 97.01 ± 0.42 SPP/PAN/PAN/SPPSPP/PAN/SPP/PAN/SPP 0.2/0.2 0.4/0.4 98.31 ± 99.860.17 ± 0.03 SPP/PAN/SPP/PAN/SPP 0.4/0.4 99.91 ± 0.04 SPP/PAN/SPPFigure5A presents the breathing resistance 0.4 at different flow rates for the97.95 monolayer ± 0.46 andSPP/PAN/PAN/SPP the multilayer structures made of a total 0.2/0.2 of 0.4 g/m2 (distributed as 97.01 0.4 g/m± 0.422 for 2 SPP/PAN/SPP/PAN/SPPthe monolayer and 0.2/0.2 g/m for the 0.2/0.2multilayer filters). The use of 98.31 the symmetric ± 0.17 multilayer structure (SPP/PAN/PAN/SPP) reduced slightly the breathing resistance at 30, 95 L/min and 160 L/min when compared to the monolayer structure. On the other hand,Figure the 5A alternate presents structure the breathing of SPP/PAN/SPP/PAN/SPP resistance at different 0.2/0.2 flow g/m rates2 had for an the overall monolayer and mildthe multilayer increase in breathing structures resistance made forof alla total the air of flows 0.4 g/m when2 compared(distributed to the as monolayer 0.4 g/m2 for the monolayerstructure. and For 0.2/0.2 the case g/m of the2 for 0.8 the g/m multilayer2 structures filters). (Figure5 B),The the use improvement of the symmetric in breathing multilayer

Nanomaterials 2021, 11, x FOR PEER REVIEW 9 of 17

structure (SPP/PAN/PAN/SPP) reduced slightly the breathing resistance at 30, 95 L/min and 160 L/min when compared to the monolayer structure. On the other hand, the alternate structure of SPP/PAN/SPP/PAN/SPP 0.2/0.2 g/m2 had an overall mild increase in breathing resistance for all the air flows when compared to the monolayer structure. For Nanomaterials 2021, 11, 900 the case of the 0.8 g/m2 structures (Figure 5B), the improvement in9 ofbreathing 17 resistance at 160 L/min in the symmetric structure was much more accused than for the 0.4 g/m2 layers. Similar to what was previously observed, the alternating structure of SPP/PAN/SPP/PAN/SPPresistance at 160 L/min in the symmetric at the higher structure basis was we muchight more did accused not improve than for the breathing resistance. 0.4 g/m2 layers. Similar to what was previously observed, the alternating structure of BasedSPP/PAN/SPP/PAN/SPP on these results, at it the can higher be basisstated weight that did filtration not improve efficiency breathing resistance.is maintained regardless of theBased disposition on these results, of the it can layers; be stated however, that ﬁltration a efﬁciencysymmetric is maintained approach regardless does result of in an improve- mentthe disposition in breathing of the layers; resistance however, compared a symmetric approachto their does monolayer result in an counterparts. improvement in breathing resistance compared to their monolayer counterparts.

Figure 5. Breathing resistance at 30, 95 and 160 L/min of monolayer spunbond polypropy- Figurelene/polyacrylonitrile/spunbond 5. Breathing resistance polypropylene at 30, 95 (SPP/PAN/SPP),and 160 L/min multilayer of monolayer spunbond spunbond polypropy- polypropylene/polyacrylonitrile/spunbondlene/polyacrylonitrile/polyacrylonitrile/spunbond polypropylene (SPP/PAN polypropylene/SPP), (SPP/PAN/PAN/SPP) multilayer spunbond and polypropylene/polyacrylonitrile/polyacrylonitrile/spunbondspunbond polypropylene/polyacrylonitrile/spunbond po polypropylene/polyacrylonitrile/spunbondlypropylene (SPP/PAN/PAN/SPP) and spunbond polypropylene (SPP/PAN/SPP/PAN/SPP) filters with a total of 0.4 (A) and 0.8 (B) basis weight polypropylene/polyacrylonitrile/spunbond polypropylene/polyacrylonitrile/spunbond polypro- (g/m2), respectively. Results are presented as the mean ± SD (n = 3). pylene (SPP/PAN/SPP/PAN/SPP) filters with a total of 0.4 (A) and 0.8 (B) basis weight (g/m2), respectively.Interestingly, Results by correlatingare presented the breathing as the mean resistance ± SD data (n with= 3). the percentage of porosity obtained in the mats (see Table2), it can be seen that the percentage of porosity is reduced when a higher basis weight is deposited, which also translates to poorer breathability.Interestingly, Therefore, symmetric by correlating splitting of the fibersbreathing nanolayer resistance is advantageous data with since the it percentage of po- rositytranslates obtained into an improvement in the mats in breathing(see Table resistance 2), it forcan the be same seen amount that ofthe SPP percentage used. of porosity is reducedThis is attributed when toa higher the breaking basis up weight of the nanofibers is deposit layer,ed, which which leads also to antranslates overall to poorer breath- increase of interfiber porosity. ability. Therefore, symmetric splitting of the fibers nanolayer is advantageous since it translates into an improvement in breathing resistance for the same amount of SPP used. This is attributed to the breaking up of the nanofibers layer, which leads to an overall increase of interfiber porosity.

Table 2. Mat porosity of the electrospun polyacrylonitrile fibers (PAN) at 0.2, 0.4 and 0.8 g/m2.

Sample Basis Weight (g/cm2) Mat Porosity (%) PAN monolayer 0.2 88.86 ± 1.06 PAN monolayer 0.4 87.90 ± 0.58 PAN monolayer 0.8 74.39 ± 2.65

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Table 2. Mat porosity of the electrospun polyacrylonitrile ﬁbers (PAN) at 0.2, 0.4 and 0.8 g/m2.

Sample Basis Weight (g/cm2) Mat Porosity (%) PAN monolayer 0.2 88.86 ± 1.06 PAN monolayer 0.4 87.90 ± 0.58 PAN monolayer 0.8 74.39 ± 2.65

3.3. Antimicrobial Activity of the Multilayer Electrospun Filters The use of inorganic particles as a multifunction agent for nanofibrous mats has emerged over the past few years as a manner to create more effective materials in the areas of biomedicine, food and filtration [22,23,27,30,41]. In this work, to generate antimicrobial nanocomposite filter materials, loadings of 1, 3, 10, and 20 wt.% of ZnO nanoparticles were added to the PAN solution and were characterized in terms of viscosity and conductivity (see Table3).

Table 3. Physicochemical properties (viscosity and conductivity) of polyacrylonitrile (PAN) and PAN

containing Zinc Oxide (PAN-ZnO) nanoparticles at 1 (PAN-ZnO1), 3 (PAN-ZnO3), 10 (PAN-ZnO10) and 20 (PAN-ZnO20) wt.%.

Sample Viscosity (cP) Conductivity (µS/cm) PAN 804.2 ± 0.95 65.98 ± 0.40 PAN ZNO1 808.9 ± 0.88 66.66 ± 0.87 PAN ZnO3 812.0 ± 3.53 69.79 ± 0.22 PAN ZNO10 924.85 ± 2.04 72.15 ± 1.54 PAN ZNO20 954.1 ± 1.11 72.89 ± 0.08

An increase in both viscosity and conductivity was observed with the increasing active filler loading. Interestingly, this increase in viscosity is not reflected in the fiber diameter. Thus, Figure6 indicates that a reduction in fiber diameter was observed with the incorporation of the ZnO. PAN without the inorganic filler had a fiber diameter of 282.5 ± 47.6 nm, whereas the ZnO-PAN nanofibers had a diameter of approximately 200 nm for all concentrations. Although there are many parameters that can influence electrospinning processing and fiber morphology [42], it may be that the driving factor to control the nanofiber size in these experiments was the conductivity. The charge difference in the solution caused by the addition of ZnO could lead to a stretching effect of the solution jet, leading to a reduction in fiber diameter. This is in good agreement with previous reports based on the addition of ZnO to PAN solutions, in which the authors suggested that the reduction in the fiber diameter was due to the change in charge density of the PAN solution with the presence of ZnO [43]. From the FE-SEM micrographs, it can also be observed that the addition of ZnO particles led to the presence of isolated beads across the morphology of the fibers (see Figure6). The presence of beaded fibers have shown benefits in air filtration, since beads can improve the permeability of the membrane and consequently lower the pressure drop [44]. Nanomaterials 2021, 11, x NanomaterialsFOR PEER REVIEW2021, 11, 900 11 of 17 11 of 17

FigureFigure 6. Field 6. emission Field emission scanning electronscanning microscope electron (FE-SEM)microscope micrographs (FE-SEM) and micrograph average fibers and diameter average of the fiber electrospun polyacrylonitrilediameter of (PAN) the electrospun and PAN containing polyacrylonitrile Zinc Oxide (PAN) nanoparticles and PAN (PAN-ZnO) containing at Zi 1nc (PAN-ZnO1), Oxide nanoparticles 3 (PAN-ZnO 3), 10 (PAN-ZnO10)(PAN-ZnO) and at 20 1(PAN-ZnO20) (PAN-ZnO1), wt.%. 3 (PAN-ZnO3), Scale bar = 10 10µ m.(PAN-ZnO10) Results are presented and 20 (PAN-ZnO20) as mean ± SD ( nwt.%.= 3). Scale bar = 10 μm. Results are presented as mean ± SD (n = 3). The biocide capacity of the ZnO-loaded nanofibers was then analyzed. The antimicro- The biocide bialcapacity effect ofof ZnOthe ZnO-loaded particles is exerted nanofibers by different was mechanisms then analyzed. of action The such antimi- as the creation of reactive oxygen species (ROS) and ion release to the fiber surface (Zn2+), which could crobial effect of ZnO particles is exerted by different mechanisms of action such as the result in membrane dysfunction, interruption and blockage of transmembrane electron 2+ creation of reactivetransport oxygen [45 species–47]. In (ROS) some cases,and ion the release antimicrobial to the performancefiber surface of (Zn ZnO), has which been reported could result in membraneto be triggered dysfunction, by humidity, interruption which is and advantageous blockage of considering transmembrane that humidity elec- will be tron transport [45–47].present In in some the environment, cases, the antimicrobial and is also created performance by the respirator of ZnO has user been during re- breathing. ported to be triggeredThis featureby humidity, also allows which the is particles advantageous to present considering a broad spectrum that humidity of inhibition will for both be present in the environment,Gram+ and G− andbacteria is also [22 crea]. ted by the respirator user during breathing. This feature also allowsIn this the regard,particles Table to4 present gathers a the broadS. aureus spectrumand E. of coli inhibitionreduction for of the both electrospun Gram+ and G− bacteriaPAN filters [22]. containing ZnO particles at different concentrations (1, 3, 10 and 20 wt.%) after 24 h. The samples were tested with the nanofibers with the base weight of 0.4 g/m2. The In this regard, Table 4 gathers the S. aureus and E. coli reduction of the electrospun filter material containing 1 wt.% of ZnO showed a strong reduction (R ≥ 3) against both PAN filters containingtypes ofZnO bacteria, particles indicating at different a strong concentrations antimicrobial (1, activity. 3, 10 and In the 20 casewt.%) of after the PAN-ZnO 2 24 h. The samplesat were 3 wt.%, tested the S. with aureus theand naE.nofibers coli showed with an the even base higher weight reduction. of 0.4 g/m For the. The sample with filter material containing3 wt.% loading 1 wt.% against of ZnOS. showed aureus, no a countsstrong werereduction measured, (R ≥ so3) byagainst default both a R > 3 was types of bacteria, considered.indicating Ona strong the other antimicr hand,obial the filters activity. containing In the ZnOcase nanoparticlesof the PAN-ZnO at 10 andat 20 wt.% 3 wt.%, the S. aureusshowed and a E. strong coli showed antimicrobial an even activity higher (R ≥ 3),reduction. but there For was the an evident sample decrease with 3 in growth wt.% loading againstinhibition S. aureus when, no compared counts were with themeasured, filters containing so by default ZnO ata R 3 wt.%.> 3 was Such con- a decrease sidered. On the othercan be hand, ascribed the with filters nanoparticle containing agglomeration ZnO nanoparticles within the at fibers10 and due 20 to wt.% excessive filler loading in the fibers. showed a strong antimicrobial activity (R ≥ 3), but there was an evident decrease in growth inhibition when compared with the filters containing ZnO at 3 wt.%. Such a decrease can be ascribed with nanoparticle agglomeration within the fibers due to excessive filler loading in the fibers.

Nanomaterials 2021, 11, x FOR PEER REVIEW 12 of 17

Nanomaterials 2021, 11, 900 Table 4. Antibacterial activity of PAN containing Zinc Oxide (PAN-ZnO) nanoparticles12 of 17at 1, 3, 10 and 20 wt.% against Staphylococcus aureus CECT240 (ATCC 6538p) and Escherichia coli CECT434 (ATCC 25922) after 24 h exposure. R corresponds to the reduction value.

Table 4. Antibacterial activity of PAN containing Zinc OxideControl (PAN-ZnO) (PAN) nanoparticlesPAN-ZnO at 1, 3, 10 and Microorganism ZnO (wt.%) R 20 wt.% against Staphylococcus aureus CECT240 (ATCCLog 6538p) (CFU/mL) and Escherichia coliLogCECT434 (CFU/mL) (ATCC 25922) after 24 h exposure. R corresponds1 to the reduction value. 4.85 ± 0.18 3.26 3 Control (PAN) PAN-ZnO No Counts R > 3 MicroorganismS. aureus ZnO (wt.%) 8.11 ± 0.23 R 10 Log (CFU/mL) Log (CFU/mL) 3.20 ± 0.20 4.91 1 20 4.85 ± 0.18 2.85 3.26± 0.14 5.26 3 No Counts R > 3 S. aureus 8.11 ± 0.23 101 3.20 ± 0.204.99 4.91± 0.12 3.02 203 2.85 ± 0.14 1.25 5.26± 0.15 6.76 E. coli 8.01 ± 0.17 1 10 4.99 ± 0.12 3.85 3.02± 0.23 4.16 3 1.25 ± 0.15 6.76 E. coli 8.01 ± 0.17 1020 3.85 ± 0.23 3.56 4.16± 0.17 4.45 20 3.56 ± 0.17 4.45 Figure 7 shows the TEM micrographs of the ZnO-PAN loaded filters, confirming the presenceFigure of7 shows the ZnO the TEMnanoparticles micrographs efficiently of the ZnO-PAN encapsulated loaded within filters, confirmingthe polymer the fibers for presenceall the tested of the ZnOZnO nanoparticles contents. For efficiently the lowe encapsulatedr ZnO concentrations within the polymer (PAN-ZnO1 fibers for alland PAN- theZnO3), tested a ZnOrelatively contents. good For dispersion the lower ZnOand dist concentrationsribution across (PAN-ZnO1 the fibers and was PAN-ZnO observed.3), How- aever, relatively as the good concentration dispersion and increases, distribution the acrosspresence the fibersof agglomerates was observed. becomes However, more pro- asnounced, the concentration especially increases, for PAN-ZnO20. the presence These of agglomerates observations becomes can be correlated more pronounced, with the reduc- especiallytion in antimicrobial for PAN-ZnO20. activity These reported observations in Table can 4, be since correlated agglomerates with the are reduction known in to reduce antimicrobial activity reported in Table4, since agglomerates are known to reduce the ef- the effective filler area, therefore diminishing their effectiveness as biocide agents [41]. fective filler area, therefore diminishing their effectiveness as biocide agents [41]. Although someAlthough degree some of agglomeration degree of agglomeration is common in nanocompositesis common in nanocomposit due to limitedes solubility due to limited ofsolubility dispersed of nanoparticles, dispersed nanoparticles, this phenomenon this mustphenomenon be avoided must as it be can avoide lead tod reducedas it can lead to performancereduced performance [27]. [27].

FigureFigure 7. 7.Transmission Transmission Electron Electron Microscope Microscope (TEM) (TEM) micrographs micrographs of polyacrylonitrile of polyacrylonitrile (PAN) and (PAN) PAN and PAN containing Zinc Oxide (PAN-ZnO) nanoparticles at 1 (PAN-ZnO1), 3 (PAN-ZnO3), 10 (PAN- containing Zinc Oxide (PAN-ZnO) nanoparticles at 1 (PAN-ZnO1), 3 (PAN-ZnO3), 10 (PAN-ZnO10) andZnO10) 20 (PAN-ZnO20) and 20 (PAN-ZnO20) wt.%. wt.%.

SimilarSimilar ﬁndings findings have have been been reported reported by other by othe authors,r authors, concluding concluding that at highthat con-at high con- centrationscentrations of of nanoparticles, nanoparticles, aggregates aggregates may may form, form, decreasing decreasing the ZnO the activity ZnO activity against against bacteriabacteria [ 28[28,48,49].,48,49]. For For this this reason, reason, the generationthe generation of multilayer of multilayer structures structures with ZnO with par- ZnO particles were prepared at a concentration of 3 wt.%, as this concentration gave optimum ticles were prepared at a concentration of 3 wt.%, as this concentration gave optimum antibacterial properties whilst being well dispersed and distributed across the ﬁbers. Con- antibacterial properties whilst being well dispersed and distributed across the fibers. Con- sidering these results, filters of PAN-ZnO3 were selected and additionally measured at

Nanomaterials 2021, 11, 900 13 of 17

sidering these results, filters of PAN-ZnO3 were selected and additionally measured at different time points of bacterial exposure (1, 3, 6, and 8 h), in order to determine the bactericidal kinetics of the nanofibers once microorganisms are stopped at the filter surface. Table5 indicates that after just one hour, a significant reduction can be seen for both types of strains, followed by a R ≥ 3 after 3, 6, and 8 h, indicating a strong bactericide behavior, and, therefore, confirming the feasibility to use these materials as potential antibacterial filters in respirators.

Table 5. Antibacterial activity of polyacrylonitrile (PAN) containing Zinc Oxide (PAN-ZnO) nanoparticles at 3 wt.% against Staphylococcus aureus CECT240 (ATCC 6538p) and Escherichia coli CECT434 (ATCC 25922) after 1, 3, 6, and 8 h exposure. R corresponds to the reduction value.

Control PAN-ZnO Microorganism Time (h) 3 R Log (CFU/mL) Log (CFU/mL) 1 6.01 ± 0.11 3.76 ± 0.21 2.25 3 6.86 ± 0.17 3.68 ± 0.15 3.18 S. aureus 6 7.18 ± 0.19 3.10 ± 0.18 4.08 8 7.86 ± 0.13 2.99 ± 0.17 4.87 1 5.98 ± 0.09 3.96 ± 0.11 2.02 3 6.36 ± 0.10 3.29 ± 0.13 3.07 E. coli 6 7.01 ± 0.14 3.32 ± 0.10 3.69 8 7.88 ± 0.11 3.28 ± 0.15 4.60

3.4. Filtration Performance of the Antimicrobial Multilayer Filters Once the antimicrobial performance of the filters was assessed, the breathing resistance and filtration efficiency were also analyzed. The biocide stacked filters were prepared as shown in Figure8A. The stacked structures contained one or two layers of PAN-ZnO 3 electrospun nanofibers with a total basis weight of 0.4 g/m2 (0.2/0.2 g/m2, see the schematics of the structures in Figure8A). The filter efficiency was similar for the three conformations, with values of 97.01 ± 0.41% for the SPP/PAN/PAN/SPP configuration, 98.36 ± 0.01% for SPP/PAN-ZnO3/PAN/SPP and 99.38 ± 0.08% for PP/PAN-ZnO3/PAN-ZnO3/SPP configuration filter, suggesting that the presence of ZnO particles in either one or two layers of nanofibers slightly increased the filtration efficiency. As per the breathing resistance (Figure8B), the presence of one or two layers of PAN-ZnO3 fibers decreased the breathability proportionally; however, the values were still acceptable if extrapolated to the area of a conventional respirator (200 cm2), and especially for the configuration of SPP/PAN/PAN-ZnO3/SPP. Factors such as small and homogeneous pore diameters and high porosity are key factors to obtain a low breathing resistance material [23,50]. In this case, the presence of nanoparticles within the electrospun fibers led to a reduction in pore size associated with a lower fiber diameter, and thus an increase in pressure drop. This is consistent with the presence of one or two layers of PAN-ZnO3 fibers, obtaining a higher breathing resistance when two split nanofiber layers of PAN-ZnO were considered (Figure8B). Nanomaterials 2021, 11, x FOR PEER REVIEW 14 of 17 Nanomaterials 2021, 11, 900 14 of 17

Figure 8. A Figure 8. (A)( Schematics) Schematics of the of themultilayer multilayer filter filter conf configurations,igurations, spunbond spunbond polypropylene/polyac- polypropy- lene/polyacrylonitrile/polyacrylonitrile/spunbond polypropylene (SPP/PAN/PAN/SPP), spunbond rylonitrile/polyacrylonitrile/spunbond polypropylene (SPP/PAN/PAN/SPP), spunbond polypro- polypropylene/polyacrylonitrile/polyacrylonitrile-zinc oxide/spunbond polypropylene (SPP/PAN- pylene/polyacrylonitrile/polyacrylonitrile-zinc oxide/spunbond polypropylene (SPP/PAN- ZnO /PAN/SPP) and spunbond polypropylene/polyacrylonitrile zinc oxide/polyacrylonitrile zinc ZnO3/PAN/SPP)3 and spunbond polypropylene/polyacrylonitrile zinc oxide/polyacrylonitrile zinc oxide/spunbond polypropylene (SPP/PAN-ZnO /PAN-ZnO /SPP). (B) Breathing resistance of the oxide/spunbond polypropylene (SPP/PAN-ZnO3/PAN-ZnO3/SPP).3 3 (B) Breathing resistance of the above-mentioned configurations, SPP/PAN/PAN/SPP, SPP/PAN-ZnO3/PAN/SPP and SPP/PAN- above-mentioned configurations, SPP/PAN/PAN/SPP,2 SPP/PAN-ZnO3/PAN/SPP and SPP/PAN- ZnO3/PAN-ZnO3/SPP with a total of 0.4 basis weight (g/m ). ZnO3/PAN-ZnO3/SPP with a total of 0.4 basis weight (g/m2). Overall, these results indicate that ZnO-loaded PAN nanofibers are an effective tech- nologyOverall, to generate these a highlyresults efficient indicate antimicrobial that ZnO-lo filteraded media PAN for respirators,nanofibers with are an values effective tech- nologyof up to to 99% generate in filter a efficiency, highly efficient and relatively antimicrob low breathingial filter resistancemedia for (approximately respirators, with values 2 of486.65–666 up to 99% Pa atin 160 filter L/min efficiency, for 50 cm andarea, relatively which would low entail breathing 120–170 resistance Pa at 160 L/min (approximately in a 200 cm2 typical respirator area) and, moreover, with a strong antibacterial activity. 486.65–666 Pa at 160 L/min for 50 cm2 area, which would entail 120–170 Pa at 160 L/min in4. a Conclusions 200 cm2 typical respirator area) and, moreover, with a strong antibacterial activity. In this study, PAN electrospun nanofibers were deposited onto non-woven polypropy- 4.lene Conclusions as monolayer fibrous structures. A range from 0.1–1 g/m2 basis weight of nanofibers was deposited, and FFP2-type filtration efficiencies were obtained when the grammage In this study, PAN electrospun nanofibers were deposited onto non-woven polypro- was above 0.4 g/m2. In order to find a balance between efficiency and breathability, dif- 2 pyleneferent configurations as monolayer based fibrous on splitting structures. and stacking A range of thefrom nanofibers 0.1–1 g/m were basis carried weight out, of nano- fiberswhich was led to deposited, improved breathingand FFP2-type resistance, filtration especially efficiencies with the symmetricwere obtained splitting when up the gram- mageof the was nanofiber above layer. 0.4 g/m To provide2. In order the filter to find with a anbalance antimicrobial between activity, efficiency ZnO particles and breathability, differentwere added configurations into the PAN solution.based on Although splitting viscosity and stacking and conductivity of the nanofibers increased were with carried out, whichthe presence led to ofimproved ZnO, both breathing filter media resistance, with and without especially ZnO with were the prepared symmetric using splitting the up of same electrospinning parameters. FE-SEM and TEM images confirmed the presence of the nanofiber layer. To provide the filter with an antimicrobial activity, ZnO particles were ZnO particles dispersed and distributed throughout the fibers, and antimicrobial testing added into the PAN solution. Although viscosity and conductivity increased with the indicated a strong bactericidal activity, especially for PAN-ZnO3, i.e., with 3 wt.% content presenceof the antimicrobial of ZnO, both filler. Infilter terms media of filtration with and performance, without theZnO presence were ofprepared ZnO particles using the same electrospinningdid not diminish filtrationparameters. efficiency, FE-SEM and althoughand TEM it increased images somewhatconfirmed the the breathing presence of ZnO particlesresistance, dispersed the values remainedand distribu lowted in accordance throughout with the what fibers, would and be acceptableantimicrobial for the testing indi- catedEuropean a strong EN149 bactericidal norm for FFP2-type activity, personal especially protective for PAN-ZnO3, equipment i.e., (PPE). with This 3 studywt.% content of demonstrates the feasibility to generate a high filtration efficiency filter media with antimi- thecrobial antimicrobial properties. It filler. also suggests In terms that of the filtration symmetric performance, splitting up of the the nanofiberpresence layer of ZnO is particles didthe optimalnot diminish method filtration to constitute efficiency, the most efficientand although filters with it increased this technology. somewhat the breathing resistance, the values remained low in accordance with what would be acceptable for the European5. Patents EN149 norm for FFP2-type personal protective equipment (PPE). This study demonstratesThe conceptualization the feasibility of this to workgenerate is based a high on the filtration patent P202030319. efficiency filter media with antimicrobial properties. It also suggests that the symmetric splitting up of the nanofiber layer is the optimal method to constitute the most efficient filters with this technology.

5. Patents The conceptualization of this work is based on the patent P202030319.

Author Contributions: M.P.-F. and A.C.-F. contributed equally and performed all experiments, measurements and data analysis. M.P.-F. wrote the manuscript draft. K.F.-L. and C.P. performed

Nanomaterials 2021, 11, 900 15 of 17

Author Contributions: M.P.-F. and A.C.-F. contributed equally and performed all experiments, measurements and data analysis. M.P.-F. wrote the manuscript draft. K.F.-L. and C.P. performed measurements and data analysis on antimicrobial studies. C.P., M.P.-F. and A.C.-F. reviewed and edited the manuscript. J.M.L. designed and supervised the research work and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work has been funded by the project “Urgent aid for the financing of scientific- innovative solutions directly related for the fight against COVID-19”, project AVI-COVID19/Nº28 funded by the Generalitat Valenciana and project Intramural CSIC 202070E104 funded by the COVID- 19 Research platform “Salud Global” from the Spanish Council for Scientific Research (CSIC). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors J.M.L., M.P.-F. and A.C.-F. are inventors in the cited patent P202030319, co-owned by Bioinicia SL and CSIC.

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