Functionalized non-woven surfaces for combating the spread of the COVID-19 pandemic

The worldwide outbreak of SARS-CoV-2 infection has necessitated mandatory use of face masks, personal protective equipment and intake of a healthy diet for immunity boosting. As per WHO's recommendation, continuous use of masks has been proven effective in decreasing the SARS-CoV-2 infection rate. The present study reports on the bacterial filtration efficacy (BFE) of a novel 4-ply functionalized non-woven face mask. We synthesized a polypropylene-based fabric with inner layers of melt-blown fine fibres coated with polylactic acid and immune-boosting herbal phytochemicals. Experimental studies on the synthesized face mask demonstrated a BFE of greater than 99% against Staphylococcus aureus (a bacterium species frequently found in mammalian respiratory tract). A thorough computational analysis using LibDock algorithm demonstrated an effective docking performance of herbal phytochemicals against harmful virus structures. More importantly, the face mask also showed sufficient and stable breathability as per regulatory standards. A breathing resistance of 30 Pa at an aerosol flow rate of 30 l h−1 was reported under standard temperature and pressure conditions, indicating a high potential for real-world applications.

PMG, 0000-0002-3993-8763; BK, 0000-0003-4257-8807 The worldwide outbreak of SARS-CoV-2 infection has necessitated mandatory use of face masks, personal protective equipment and intake of a healthy diet for immunity boosting. As per WHO's recommendation, continuous use of masks has been proven effective in decreasing the SARS-CoV-2 infection rate. The present study reports on the bacterial filtration efficacy (BFE) of a novel 4-ply functionalized non-woven face mask. We synthesized a polypropylene-based fabric with inner layers of melt-blown fine fibres coated with polylactic acid and immune-boosting herbal phytochemicals. Experimental studies on the synthesized face mask demonstrated a BFE of greater than 99% against Staphylococcus aureus (a bacterium species frequently found in mammalian respiratory tract). A thorough computational analysis using LibDock algorithm demonstrated an effective docking performance of herbal phytochemicals against harmful virus structures. More importantly, the face mask also showed sufficient and stable breathability as per regulatory standards. A breathing resistance of 30 Pa at an aerosol flow rate of 30 l h −1 was reported under standard temperature and pressure conditions, indicating a high potential for real-world applications.

Introduction
An outburst of contagious SARS-CoV-2 pandemic resulted in spread of infection waves, radically affecting health conditions of people across the world and leaving behind long-term respiratory health effects [1]. As per WHO recommendations, the spread of viral infections can be restricted with continuous use of protective masks and personal protective equipment suits [2], since the viral infections mainly occur through aerosol droplets in air which directly attack the human respiratory system [3,4]. Non-woven face masks are widely used due to tailorable fibre diameters for various layers, filtration of aerosol particles, sterility, low production costs, shorter production cycles, leverage to incorporate antibacterial materials and higher flexibility [5][6][7][8][9]. Considering various non-woven fabric manufacturing techniques, a combination of melt-blown and spun-bonded non-woven fabrics shows stable air filtration properties for biomedical applications [10].
Tellier et al. [11] reported that the spread of virus occurs rapidly through respiratory tract due to the presence of aerosol particles in the air. However, the transport of these infections aerosols can be mitigated by applying a barrier prior to their entry to the respiratory tract [12]. Hence, continuous use of face masks became an important precautionary measure for protection against deadly infectious viruses [13]. This necessitated modifications in the morphology of the protective masks to achieve a bacterial filtration efficacy (BFE) of 99% (or higher), leading to development of the multiplelayered structure exhibiting ameliorated filtration performance [14]. In a recent study [15], we demonstrated that the incorporation of polylactic acid (PLA) and traditional Indian phytochemical-based filtration layers in cotton-based face masks renders an immuno-boosting effect to the mask wearer and enhances BFE of the protective mask [16]. It has been identified that factors such as the surface area of aerosol particles and surface wettability of non-woven surfaces play a key role in the transmission of virus from the environment into human body [17]. It has also been established that hydrophobic surfaces restrict the passage of aerosol particles through the non-woven surfaces, whereas hydrophilic surfaces permit absorption of aerosol particles [18].
Based on the investigations of Ayurveda, phytochemicals or oil extracts of spices demonstrate inflammatory response to counter the biological harmful viral infections [19][20][21]. In a recent study, Xiong et al. [22] reported the effectiveness of Chinese herbal-based extracts against the harmful SARS-CoV-2 viral infection. It was also observed that glucocorticoids-based drug treatments for SARS-CoV-2 infections showed decline in the mechanical ventilation and mortality ratio among the affected patients when compared with standard care protocols [23]. Bartonkova et al. [24] reported the effective performance of herbal extracts on glucocorticoid based receptors, which exhibited anti-hypertensive, anti-fungal, anti-microbial and anti-inflammatory properties, boosting the immune response against the harmful viral infections. Similarly, Kumar et al. [25] demonstrated non-mutagenic, non-toxic, non-irritant and biodegradable performance of organic compounds at a lower concentration for plant-based herbal phytochemical oils.
The present study reports on the BFE of a functionalized 4-ply non-woven face mask and its immune-boosting characteristics. The functionalization of non-woven substrates was achieved by spray coating of polymeric layer over the meltspun non-woven filter media. This was followed by performing a thorough computational analysis using LibDock algorithm to analyse the docking performance of herbal phytochemicals against infectious virus structures. Field-emission scanning electron microscopy (FE-SEM) was performed on the functionalized fibres to examine the compact microstructure and to identify the impact of fibre augmentation on aerosol penetration efficacy. Subsequently, the surface wetting analysis of the spray-coated non-woven layer was investigated using a contact angle goniometer. Finally, the developed face mask was tested against Staphylococcus aureus (ATCC 6538, an international standard testing strain for disinfectants) to examine the BFE of the augmented 4-ply mask. The results of this study are therefore expected to be of high value for the scientific and engineering community.

Fabrication of the mask material and surface morphology
A four-ply non-woven breathable face mask is shown in figure 1a,b. The hydrophobic outer layer consists of nonwoven spun-bonded polypropylene fibres exhibiting a uniformly distributed diameter of 15.87 ± 0.5 µm figure 2b, which renders a compact close-packed structure, and improved surface area for restricting the transmission of aerosol droplets (figure 2a-f ) [26,27] . Finely distributed non-woven fibres (less than 17 µm), and thickness of 0.57 mm, further support in obstructing the entry of aerosol particles (50-500 nm) for improved filtration efficacy [28]. The second layer consists of non-woven melt-blown fine fibres exhibiting a diameter of 613.3 nm (figure 2d), which was used as filter media in the face mask. As depicted in figure 2d, a dense structure of uniformly distributed melt-blown fibres could potentially aid in enhancing the bacterial and viral filtration performance of the face mask [29,30]. Third layer of the mask was fabricated using spray coating of herbal phytochemical encapsulated PLA. This was performed to enhance the immune-boosting characteristics via inhalation to the mask wearer [31][32][33]. Mean thicknesses of the outer to inner layer (four layers) of the mask were found to be 0.28 ± 0.01 mm, 0.13 ± 0.01 mm, 0.13 ± 0.01 mm and 0.21 ± 0.01 mm, respectively. The thickness of entire face mask was found to be 1.25 ± 0.01 mm.

Bacterial filtration efficiency and breathing resistance
It is established that spun-bonded polypropylene fabric layers facilitate the removal of large aerosol particles and particulate matter (PM), whereas the lower sized molecules get trapped in compact melt-blown polyester-based filter media and PLA/herbal-extract spray-coated layer [34]. FE-SEM micrographs (figure 2a-f ) demonstrate densely packed fibre morphology of the non-woven filter layer, with uniform fibre diameter distribution. It has been established that a uniformly distributed fibre diameter facilitates narrow pore sites for trapping the aerosol and PM particles and enhances the active surface area [35,36]. Thus, the high BFE of the mask's filter media can be linked to the fibre diameters. The stable air filtration ability of the filter layer was attributed to the adsorption phenomena of the small sized impurities as per royalsocietypublishing.org/journal/rsfs Interface Focus 12: 20210040 the following five mechanisms: (i) gravity effect, (ii) inertial impaction, (iii) electrostatic effect, (iv) interception and (v) Brownian diffusion [37]. These mechanisms might show variations in filtration capabilities of fibre layers, whereas the overall filtration performance of protective mask was determined by the collective performance of several mechanisms considering the particle size of particulate impurities in atmosphere [38,39]. The addition of herbal-extract phytochemicals in layered structure of protective mask facilitated enhanced filtration efficiency and also it did not affect the differential pressure, thereby improving the overall bacterial protection against viral infections and harmful pathogens [40]. In the present investigation, four-layered non-woven protective mask with herbal phytochemicals demonstrated BFE of 99.65% against Staphylococcus aureus bacteria (as per AATCC 6538 standard), with an average bacterial count as 10 and average bacterial count for positive control as 2900. Note that the BFE of the mask without herbal phytochemicals was found to be 95% as per the abovementioned test standards. Thus, it was observed that the inclusion of herbal phytochemicals helped in enhancing the BFE of the face mask. This advancement was further acknowledged by the regulatory agencies. The developed non-woven protective mask passed European Commission's regulatory certification (Certificate No. 17020). The face mask is currently registered with a trademark name of Aushadatara Trademark No. 4595855. The fibre diameter distribution analysis is presented in figure 3a-f, and its main results are tabulated in table 1. As depicted, it was found that the outer layer, and spraycoated layers (both washed and unwashed) exhibited constant fibre diameter in the range of 17-18 µm, whereas the inner melt-blown layer (washed and unwashed) revealed fine fibre diameter ranging from 0.731 µm to 1.1 µm. Fibre distribution analysis showed that all layers possess uniform fibre diameters, thereby confirming the consistency of face mask fabrication process.
As per Indian IS 9734:2002 standard, the protective mask demonstrated low differential pressure and breathing resistance across layers of protective mask. Details on the breathing resistance of the face mask are reported in table 2.

Computational analysis and immune-boosting characteristics
The utilization of herbal phytochemicals was beneficial for imparting antibacterial characteristics in the developed mask. An investigation by Ali et al. [41] demonstrated the antibacterial performance of Azadirachta indica (A. indica) against bacteria by developing an inhibition zone of 14.5 mm, which was attributed to the presence of nearly 140 biologically active constituents. They argued that the existence of several bioactive compounds like triterpinoids, alkaloids, asteroids, reducing sugars, flavonoids, tannins, phenolic compounds and sesquiterpene lactones collectively imparted antibacterial activity [41]. Consequently, A. indica has been used in treating various skin infections, dental problems and skin inflammations [41].     royalsocietypublishing.org/journal/rsfs Interface Focus 12: 20210040 molecular interactions with targeted molecules [44]. Similarly, Balasubramanian & Kodam [45] reported herbal lavender oil functionalized PAN nanofibres which exhibited antibacterial resistance against S. aureus with K. pneumoniae with a zone of inhibition of 14-15 mm. Considering the results discussed in above-mentioned studies, herbal phytochemicals were found to be safe in lower concentration for immunity boosting, traditional home remedies and personal hygiene. Diffusion of these herbal phytochemicals in low concentration through fibre layers and subsequent inhalation helps in improving the immunity boosting, personal hygiene, and gives comfort feeling, e.g. Vicks inhalers, to the mask bearing person [46]. Computational analysis of the phytochemicals as per formulation (A) for countering protein-structured viral and bacterial targets is significant for predicting the performance of constituent phytochemicals for impeding the virus protein structure [47] (figure 4). Computational investigation of phytochemicals was performed with scoring function based on LibDock algorithm. An in silico analysis predicted the performance of structural characteristics of phytochemicals and protein-ligand complex for countering multiple bacterial targets. Docking score determines the structural complementarity of phytochemicals for analysing the neutralizing characteristics of phytochemicals against protein structures. Neutralizing performance of phytochemicals is accredited to their excellent docking score and capacity to undertake an interaction for protein unfolding. Higher docking score of salanin, arachidic acid, stearic acid and palmitic acid to counter protein structure of SARS-2 virus was attributed to the existence of hydrogen bond acceptor and aliphatic chains as it amplifies hydrogen bonding within protein-ligand compound due to attraction between electron lone pair and acceptor positive charge [48]. Aliphatic structure leads to efficient spin of bonds for occupying the targeted binding location, thereby improving the docking performance of constituent chemicals for countering the protein structure [48,49]. Neutralizing performance of stearic acid to counter the protein folding of H7N9 virus was enabled due to the presence of hydrogen bond acceptors. Similarly, the confined performance of cis-11-octadecanoic acid and arachidic acid to counter the protein structure of Dengue virus was attributed to the aliphatic structure of constituent herbal extracts, leading to an efficient-binding performance at targeted protein interface. Structural compatibility of salanin against influenza virus demonstrated an optimal docking score, resulting in improved binding of ligand structure against the protein-structured binding sites. Hindered performance of oleic acid and stearic acid was significant due to structural compatibility of constituent chemicals to counter hantavirus. Structural attributes of palmitic acid, stearic acid and oleic acid against hepatitis C virus demonstrated an enhanced docking score leading to improved binding of ligand molecules across the protein structures. The neutralizing performance of oleic acid and linoleic acid was significant for exhibiting structural compatibility against Ebola virus and herpes simplex virus. Computational analysis revealed antibacterial/microbial performance of ayurvedic phytochemicals to counter the protein-structured virus, for restricting the spread of viral and bacterial infections through COVID-19 pandemic.
The heat map analysis of constituents (as shown in figure 5) illustrates the molecular binding characteristics of formulation (B) for denaturing the virus's cell wall and/or membrane. Individual docking performance of constituent chemicals as per the high energy of the Ludi algorithm elucidates promising bioactivity of salanin, nimbin, gedunin and mahmoodin for inhibiting the potent effect of influenza A virus. The structural compatibility of the formulation was dominated by pharmacophore of chemical constituents which was more compatible with the active binding sites of influenza Avirus. Similarly, molecular docking performance of salanin, stearic acid and oleic acid contribute with mild to moderate binding against the homology modelled envelope protein of SARS-CoV-2. The structural docking was attributed to a combined effect of more hydrogen acceptors and rotation of bonds in aliphatic structures of stearic acid and oleic acid. Furthermore, constituent chemicals of A. indica, S. aromaticum, Santalum album and Ocimum tenuiflorum exhibited potent behaviour against the protein-structured cell wall (except glycoprotein of herpes virus). Among herbalextract major constituents, the bioactivities of stearic acid, linoleic acid, oleic acid, palmitic acid, nimbin and nimbolin were found to be more effective for inhibiting the activity of airborne viruses. Availability of hydrogen bond acceptors in nimbin and nimbolin elevates the extent of molecular docking, whereas aliphatic characteristics and relatively low molecular polar surface areas of oleic acid, linoleic acid, stearic acid and palmitic acid facilitate the structural combability against the protein structures of viruses. The herpes virus wrapping of glycoprotein D was dominated by chemical constituents of Eucalyptus citriodora and S. aromaticum due to the optimized energy per unit of the Ludi algorithm of eugenol, o-cymene and limonene.

Contact angle analysis
The surface wetting analysis of spray-coated C6-fluorocarbon on polypropylene-based non-woven layer against water molecules was investigated using a contact angle goniometer. Fluoro molecule-dispersed solution of C6-fluorocarbon (TUBIGUARD 30-F) was spray coated [50] on the non-woven material in such a way that it retained the porous structure of the non-woven fabric and formed a thin layer of 0.1 µm on fibre surface. C6-Fluorocarbon includes six carbon-based molecules in its main structure, bonded to different fluorine atoms. CHT Group (from Germany) have demonstrated textile finishing using C6-based fluorocarbon for decreasing the surface energy of spray-coated non-woven surface, for enhancing the water repellency [51][52][53][54]. Static contact angle of spraycoated C6-fluorocarbon on polypropylene non-woven layer was found to be 130 ± 2°(outer layer of face mask) and 91.1 ± 2°, respectively ( figure 6a,b). CHT Group further investigated the activity of C6 fluorocarbon for inhibiting the bacterial and viral contamination due to adsorption of aerosol droplets over the exposed layer of the protective mask [54].

Bacterial filtration efficiency and anti-microbial characteristics
BFE of the face mask was evaluated as per ASTM-F2101-19 standard [48,55]. A prepared test specimen was incubated at a temperature of 21 ± 5°C and relative humidity around 85% ± 5% for 4 h in order to adapt adequate operating conditions as per described in practice E171/E171M. The bacterial culture was prepared using adequate volume of tryptic soy broth, conditioned at 37 ± 2°C for duration of 24 ± 2 h in the presence of mild shaking conditions. The developed bacterial culture was diluted in peptone water for achieving concentration of 5 × 10 5 CFU ml −1 . The bacterial challenge delivery rate was adjusted to 1700-3000 viable particles per test. The flow rates of the bacterial challenge were monitored using a six-stage viable particle cascade impactor, without any test specimen to evaluate the effectiveness of aerosol particles during the testing. The aerosol particles were directed towards mask material, and the bacterial challenge was delivered to nebulizer using a peristaltic pump and further exposed on the cascade impactor. Mean particle size of the aerosol droplets was adjusted at 3 ± 0.3 µm. The test specimen was mounted on the agar plates and kept in cascade impactor such that outer layer faced the flow of aerosol particles. The aerosol particles were exposed over the test specimen for a duration of 2 min, with flow rate adjusted to 28.3 l m −1 . After the completion of positive control of test specimen, agar plates were removed and conditioned at 37 ± 2°C for a time duration of 48 ± 2 h. The negative control was obtained by collecting a sample of air on agar plates from specimen chamber after the duration of 2 min and incubated. The agar plates were counted for bacterial growth, and BFE of the test specimen mask was determined by the following equation: bacterial filtration efficiency (%)

Field-emission scanning electron microscopy
Surface morphology of the non-woven protective face mask was investigated by using FE-SEM (Model: FE-SEM, Make: Carl-Zeiss AG, JSM-6700F, Germany). The surface morphology of fine fibre structures in non-woven fabrics was analysed for assessing the compactness in surface area of protective face mask. Surface morphology analysis was important for understanding the filtration characteristics of non-woven layers.

Contact angle analysis
Surface wetting analysis is important for understanding the interaction of liquid-phased aerosol particles in air with mask material [57]. In the present study, a drop shape analyser (Model: DSA 25E, Make: Kruss GmbH, Germany) was used for the contact angle analysis. WCA was recorded by placing a standard 8 µl drop of deionized water on the surface at room temperature of 25 ± 2°C, and the contact angle was measured using Young-Laplace method [15]. WCA was recorded four times, and the final average value was taken as standard.

Computational analysis of phytochemicals
Computational assessment of two separate formulations A and B (details given in §7.2) of phytochemicals to counter the bacterial and viral protein structures was performed using docking scorefunctioned LibDock algorithm [58,59]. The algorithm was functioned for docking ligands at active receptor location, supporting ligand configurations to polar and non-polar receptor interactions. Recording of the optimized scoring positions, and the process mainly depends on binding energy, rotation of bonds and structural properties [60]. The docking performance of interactions was improved by Broyden Fletcher Goldfarb Shanno algorithm along with steady force field minimization. Heat map technique was more precise compared to picto-trendline or dot map for determining the spread of infectious bacteria and virus [61]. Proteins and ligands at drug target site were retrieved from protein data-bank (PDB) structure database. Theoretical models of ligand and protein structures were used to record the docking score. Main phytochemical constituents of the therapeutic Santalum album, A. indica, Cinnamomum camphora, Eucalyptus citriodora, Ocimum tenuiflorum, S. aromaticum and C. longa L. were sourced from PubChem database. Raw protein structures were obtained from PDB database. Docking analysis was performed to investigate improved binding activity of constituent phytochemicals by insertion of missing atoms in incomplete residues and modelling of missing loop areas, removing alternative conformations, standardizing the atom names and removing destructive steric clashes. The molecular interactions within ligand and receptor were recorded by analysing molecular docking performance, using computer-aided drug design and structural molecular biology.
Data accessibility. This article does not contain any additional data. Authors' contributions. N.A.P. and P.M.G. performed writing, reviewing and editing of the manuscript, along with data curation and investigation. D.S. performed computational analysis, and its results and discussion. H.P. and M.K. performed the industrial scale-up trials, and regulatory testing of the product formulations. B.K. spearheaded the project conceptualization initiative, monitored experimental activities and supervised the whole project.