Biosynthesis of Silver Nanoparticles Enhanced Antibacterial Silk Face Covering

ABSTRACT Biosynthesis of silver nanoparticles (AgNPs) using plant extracts as reducing agents has recently attracted interest as an environmentally friendly and cost-effective approach. In this study, biosynthesized silver nanoparticles were coated on cotton fabric as an antimicrobial outer middle layer in a silk face mask. The AgNPs were synthesized and characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). A solution of 100 ppm AgNPs was coated on cotton fabric by the exhaustion process and quantified according to the procedures of AATCC 100–1999. Top performance characteristics of the barrier face covering were determined according to ASTM F3502–2021. The AgNPs were spherical with particle size ranging 35–88 nm. SEM images of AgNP-coated cotton fabrics showed good particle dispersion that decreased after washing, with 99% reduction in viable E. coli and S. aureus after 30 washing cycles. The developed silk face covering also offered sufficient sub-micron particle filtration efficiency and airflow resistance as level II performance under ASTM classification.


Introduction
During the COVID-19 pandemic, N95, FFP2, and FFP3 respirators and medical masks were highly recommended for healthcare workers, with non-medical masks suggested for the general public (World Health Organization 2020).The filtration and particle capture mechanisms of N95 respirators and surgical masks filter out smaller particulate matter, bacteria, and viruses (Adanur and Jayswal 2020).Surgical masks and N95 respirators are usually made from thermoplastic nonwoven fabrics, with the designs intended as a protective barrier providing fluid resistance.Non-medical or homemade masks, such as scarves or bandanas do not filter aerosol sizes between 30 and 600 nm but can capture large respiratory droplets, which are a significant mode of transmission of SRS-CoV-2 (Hao et al. 2020).These masks for public use are normally made from easily and inexpensive materials, such as cotton, silk, linen, and wool.Multi-layered structures of woven fabric can exceed the efficiency of materials used in some medical face masks (Zhao et al. 2020).These fabric masks have low filtration efficiency and retain moisture, favoring bacterial or viral organisms that might cause risk of infection (MacIntyre et al. 2015).Pore size of fabric masks expands after rubbing, with damage to the sample caused by abrasion.Therefore, fabric masks are not recommended for healthcare workers (Davies et al. 2013).
The new standard "ASTM F3502 standard specification for barrier face coverings" was established in response to the global COVID-19 pandemic to describe a product that was neither a medical mask nor an inhalation protection respirator.Principal performance criteria for face covering only emphasized sub-micron particle filtration efficiency and airflow resistance (American Society for Testing and Materials 2021).Particulate filtration efficiency was quantified as the ability of the face covering to remove aerosol particles passing through the material, while airflow resistance properties indicated mask comfort and breathability.Airflow resistance could be measured by differential pressure.The higher the differential pressure, the harder it was to breathe when wearing the mask (Forouzandeh, O'Dowd, and Pillai 2021).Lima et al. (2022) also suggested that an airflow resistance value of the respirator above 8 mm H 2 O was considered unbreathable.Compared with medical surgical masks and N95, FFP2, or FFP3 respirators, this new standard of face coverings also reduced the risk of virus infection and effectively prevented pathogens from spreading in the environment.Face coverings that meet the ASTM F3502 requirement could be used by the general public.Functional properties such as antimicrobial (Kharaghani et al. 2018;Li et al. 2006) and water repellence (Ray et al. 2020) could also be added to the face covering.
Silk is a decent material for fabric masks; it had a high hydrophobic barrier to droplets and was breathable and reusable after cleaning (Parlin et al. 2020).Four layers of silk fabric protected against particulate penetration ranging from 10 nm to 6 µm (Konda et al. 2020), while the silk filament showed high rubbing resistance, low chance of pilling, excellent antimicrobial property, and good air permeability (Kaur et al. 2014;Phoophat et al. 2020).Eri silk, a protein fiber derived from cocoons made by wild silk moths, also showed the unique characteristics of lightness, soft-smooth feel, thermal property, and high tensile strength (Chollakup and Smitthipong 2012;Rungruangkitkrai et al. 2020).
An antimicrobial textile inhibited the growth of bacterial infections through the material (Abou Elmaaty et al. 2022).However, the protective layer of the face mask was not able to filler microorganisms, which may cause a health risk to the user (Hiragond et al. 2018).Silver nanoparticles (AgNPs) had been used to produce antimicrobial textiles because of their antibacterial, antifungal, anticancer, anti-inflammatory and UV protection properties (Emam, El-Rafie, and Rehan 2021;Syafiuddin 2019).Biosynthesis of AgNPs was suggested as an alternative method to enhance the antibacterial properties of the face covering and replace chemical and physical processes.This method was more environmentally friendly and cost-effective with reduced use of chemicals (Yaqoob, Umar, and Ibrahim 2020).Previous research reported the utilization of basil (Brahmachari et al. 2014), banana leaves (Ibrahim 2015), banana peel (Bankar et al. 2010) and sweet flag root (Nakkala et al. 2015) to biosynthesize AgNPs that can inhibit microorganisms at lower concentrations than other heavy metals.Moreover, some natural polymers such as chitosan (Hasan et al. 2022;Mohammed, Hassan, and Hassan 2023), pectin (Zhao et al. 2022), xanthan gum (Emam and Zahran 2015), acacia gum (Emam et al. 2015), gum ghatti and gum olibanum (Kora and Sashidhar 2015) were used as the capping and/or reduction of AgNPs in the green synthesis method.In addition, small amounts of AgNPs do not harm eukaryotic cells while destroying bacterial cells in the human body.
This study synthesized AgNPs using green technology.The AgNPs were coated on cotton fabric as an antibacterial layer in the silk face covering.The antimicrobial activities of AgNPs and AgNPs coated on cotton fabric were analyzed by an antibacterial assay.The performance of the face covering for submicron particle filtration efficiency and airflow resistance was also evaluated according to the ASTM standard requirement.

Materials
Mangosteen (Garcinia mangostana Linn.) peels were purchased from a local grocery store in Bangkok, Thailand.Silk filament fabric 91.62 g/m 2 and Eri silk fabric 74.29 g/m 2 to make the face covering prototype were obtained from Natural Niche Co., Ltd.Silver nitrate (AgNO 3 ) was purchased from Sigma Aldrich.A water-repellent agent, namely perfluorooctanoic acid-free fluorocarbon (PFOA-free fluorocarbon) was purchased from Star Tech Chemical Co., Ltd.(Bangkok, Thailand).

Preparation of peel extract
To make the aqueous mangosteen peel extract, the fruit peels were first washed with deionized water, then cut into small pieces and dried in a hot air oven at 60°C until moisture content was less than 8%.The dry mangosteen peels were then ground to a coarse powder and 20 g was boiled in 100 ml of double distilled water for 30 min while stirring occasionally.The aqueous extract was cooled, filtered using Whatman No.1 filter paper and stored at 4°C for further use to synthesize AgNPs from an AgNO 3 precursor solution.

Silver nanoparticles synthesis
Synthesis of AgNPs was carried out as described previously (Aritonang, Koleangan, and Wuntu 2019).Briefly, the aqueous extract of mangosteen peel was added to 10 ml of AgNO 3 solution.One milliliter of 20% plant extract was added to 50 ml of AgNO 3 solution, and the mixture was stirred continuously for 3 h at 90°C.In each reaction vessel, color changed to yellowish brown (Selvakumar et al. 2012).Furthermore, the mixture was stored at 4°C for the antibacterial activity test and analyzed by using UV -Vis spectrophotometer.The AgNPs were characterized by FT -IR, EDS, FESEM.

Face covering design
According to ASTM F3502-21, design criteria of face barrier coverings included setting minimum areas of face coverage over the wearer's nose and mouth.In this study, the pattern of the face covering was designed following previous research that suggested the lookalike cup mask was more suitable and comfortable for the wearer (Morishima and Mitsuno 2019).PFOA-free fluorocarbon at 2% concentration by the pad-dry-cure method was coated on the filament silk fabric (outer layer of face covering), as suggested by the minimum amount in the leaflet sheet and previous studies (Chowdhury 2018;Srichola et al. 2022).Figure 1 shows that the outermost hydrophobic layer was made of filament silk fabric (91.62 g/m 2 ) coated with PFOA-free fluorocarbon to improve the hydrophobic property of silk.So the water contact angle of the outermost layer was approxiamtely 133.44 ± 2.87°.The two middle filter layers comprised AgNPs coated on cotton fabric (76.61 g/m 2 ) and spunbond polypropylene nonwoven fabric to improve filtration efficiency of the silk face covering.The innermost layer was made of pristine eri silk fabric (74.29 g/m 2 ).

Coating of face covering with AgNPs
The outer middle layer of the face covering performed as the filtration medium and the antimicrobial layer as can be seen in Figure 1.Scoured cotton fabric (76.61 g/m 2 ) was coated with AgNPs by the exhausting method.An AgNPs solution (100 ppm) with 2 g/l dispersing agent was prepared following (Jha and Prasad 2016).The process was carried out at 100°C for 30 min (L:R = 1:15).Then, cotton fabric coated with AgNPs was rinsed and dried at room temperature and used to produce the outer middle layer of the silk face covering.To evaluate the durability of the AgNP-coated cotton fabric, ISO 6330:2012 was applied for 30 washing cycles.

UV-visible spectroscopy
The resultant nanopowder from each of the reactions was re-suspended in an equal amount of sterile de-ionized water and spectrum cectrophotometer UV-1280 in wavelength range 200-800 nm.

Fourier transform infrared spectroscopy
Fourier transform infrared (FT-IR) spectroscopy was used to establish the identity of the phytochemical constituents involved in the reduction and stabilization of the silver nanoparticles.An FT-IR spectrum for powdered AgNPs was obtained using a Perkin Elmer FT-IR Spectrophotometer Frontier following the attenuated total reflectance (ATR) technique in the range 4000-500 cm −1 .The presence of AgNPs coated on the cotton fabric was investigated using a Thermo Scientific Nicolet IR200.

Scanning electron microscopy
Surface morphology, particle size, and composition of the AgNPs were investigated by field emission scanning electron microscopy (FESEM) using a Hitachi model, Jeol JSM-5600 LV with accelerating voltage of 2 kV and energy-dispersive X-ray spectroscopy (EDX, Zeiss Supra 35VP).Morphology, qualitative, and quantitative elemental composition of AgNPs on the cotton fabric were characterized by scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX).

Antibacterial activity evaluation of cotton fabric
Evaluation of the antibacterial properties of the AgNP-coated cotton fabrics was conducted quantitatively according to AATCC 100-2019.S. aureus and E. coli were used as the gram positive and gramnegative organisms, respectively, according to the reference bacterial strains (Sathianarayanan et al. 2010).This test was performed on pristine cotton fabric and AgNP-coated fabric with contact time 18 h.Bacterial percentage reduction was calculated using formula (1): where R = percentage reduction, A = number of bacteria recovered from inoculated treated test specimen swatches in the bottle incubated over the desired contact time and B = number of bacteria recovered from inoculated untreated test specimen swatches in the bottle incubated over the desired contact time.

Performance of the silk face covering
The performance of the developed face covering was tested according to ASTM F3502-21; sub-micron particulate filtration efficiency and airflow resistance were tested by TSI model 3082.

Synthesis and characterization of the silver nanoparticles
In this study, synthesis of silver nanoparticles using G. mangostana peel extract as the reducing agent under optimal conditions was viewed after 3 h by color change from light yellowish to dark brown, as shown in Figure 2  The FESEM images at a magnification 15,000 revealed small spherical grains of AgNPs (Figure 4).At a magnification of 45,000, most AgNPs in all mixtures exhibited round shapes as same as previous study (Hawar et al. 2022) with average size 40 nm (range 35-88 nm).Size particle distribution of AgNPs is shown in Figure 4, which was classified mostly (>50%) in range 21-50 micron.
Figure 5 shows the FT-IR spectra of synthesized AgNPs from G. mangostana peel extract after reaction with AgNO 3 .FT-IR measurement based on AgNPs mediated by G. mangostana peel extract revealed different absorption peaks at 3424,2969,2904,2837,2098,1640,1384,1197,1158,1117,1039,919,827,620,594,542,416, and 408 cm −1 .For AgNPs, a very strong absorption peak shifted toward a lower wave number was observed at 3424 cm −1 , indicating binding of Ag+ with hydroxyl and or amine groups in the G. mangostana peel extract interacted with the surface of the AgNPs as same as the previous research (Khane et al. 2022).Other bands at 2969, 2904, and 2837 cm −1 were assigned to stretching vibration of the hydrocarbon (C -H) bond of alkenes, while the prominent peak at 1640 cm −1 indicated involvement of amide -I bond (-C=O) of proteins as a capping agent and stabilization of  The peak at 1384 cm −1 was assigned to C -H symmetric vibrations and also observed in G. mangostana peel extract (Nishanthi, Malathi, and Palani 2019).

Analysis of AgNP-coated cotton fabric
Surface morphology and the 3-dimensional nature of AgNP-coated cotton fabric (CE100) and AgNP-coated cotton fabric after 30 washing cycles (CE100-30W) were examined by SEM, with results shown in Figure 6.The SEM image of AgNP-coated cotton fabric showed clusters of silver particles concentrated in some areas of the fabric.After exposure to 30 consecutive washing cycles, the clusters of silver particles decreased and dispersed on the coated fabric surface.SEM equipped with EDX employed to record the SEM images and analyzed the elemental composition of silver, as shown in Figure 7. Quantitative analysis of the silver content is shown in Table 1.
It was found the EDX spectrum of some basic elements, such as carbon and oxygen (Figure 7).The peak at 3 keV confirmed the presence of silver elements as they appeared in both AgNP-coated cotton fabric and the washed fabric samples.The elemental composition showed the presence of 3.13 and 1.52 atomic percentage of Ag at selected areas for AgNP-coated cotton fabric and AgNP-coated fabric after 30 washing cycles.This indicated that Ag content of AgNP-coated cotton fabric only slightly decreased after the washing cycles because the AgNPs were chemically bonded on the surface of the fabric (Zhang et al. 2013).

2016
). Peaks at 3333-3334 cm −1 were assigned to O -H stretching vibration, while peaks at 2896 cm −1 or 2899 cm −1 and 1361 cm −1 and 1369 cm −1 were assigned to C -H stretching and C -H bending, respectively.The peak at 1029 cm −1 was related to C -O stretching, while peaks at 3334 cm −1 and 1012 cm −1 or 1018 cm −1 were related to O -H stretching and O -H bending, respectively.However, the FT -IR spectrum of CE100-30W was slightly different from the uncoated cotton.These results suggested that the AgNPs were chemically bonded with hydroxyl groups in cellulose macromolecules of the cotton fabric (Baruah 2016).

Antibacterial activity of AgNP-treated fabric
The uncoated cotton fabric, CE100 and CE100-30W were challenged with E. coli and S. aureus.Viable bacterial counts recovered from the fabrics before and after incubation are shown in Table 2.After 18 h of incubation, the AgNP-coated fabric showed percentage reduction in viable E. coli and S. aureus at 99.92% and 99.94%, respectively.After 30 washing cycles, the antibacterial activity did not significantly decrease, with percentage reductions in viable E. coli and S. aureus 99.96% and 99.96%.So the washing process was slightly affected to the antimicrobial properties similar to some previous studies (Aladpoosh, Montazer, and Samadi 2014;Maghsoudi et al. 2022;Xu et al. 2017).This indicated that the AgNPs were  fixed on cotton fabric surface because of electrostatic attractions between oppositely charged AgNPs and the surface of the cotton fiber (Zhang et al. 2013).It could be referred that this face covering mask was durability enough as it could be used more than 30 times with the retained antibacterial activity.A clinical test in mice at a dose of 2000 mg/kg AgNPs resulted in no death or injury (Kim et al. 2013).Thus, this green technique of AgNP biosynthesis can be applied as a cotton fabric filter in the outer middle layer of the face covering to add antibacterial property.

Sub-micron particle filtration efficiency and airflow resistance
The principal performance properties of the developed silk barrier face covering are shown in Table 3. Sub-micron particulate filtration efficiency was 67.98%, which met the requirement of Level II of ASTM F3502 standard but was not comparable to the N95 respirator, which was at least 95% (Forouzandeh, O'Dowd, and Pillai 2021).This silk barrier was effective for public use during the COVID-19 pandemic.However, Skaria and Smaldone (2014) found that the airflow pressure of the three-layer surgical mask and N95 respirator was about 1.87 and 2.67 mm H 2 O respectively.So this developed face covering might be less comfort to wear than the three-layer surgical mask and N95 respirator according to this face covering compiled with four layers.However, the developed silk face covering was comfortable enough for breathing and suitable to wear on a daily basis.

Conclusions
The outer middle layer of the silk face covering consisting of biosynthetic AgNPs coated on cotton fabric enhanced antimicrobial properties and filtration efficiency.Silver nanoparticlecoated cotton fabrics showed good particle dispersion, while percentage reduction of E. coli and S. aureus did not change after 30 washing cycles.Silk face coverings with AgNP-coated cotton fabric as the outer middle layer, filament silk fabric as the outermost layer and eri silk fabric as the innermost layer offered acceptable sub-micron particle filtration efficiency and airflow resistance at 67.98% and 2.87 mm H 2 O, respectively.These values met the requirement of ASTM classification for barrier face coverings at level II performance.Further studies should focus on the durability of this silk face covering as an important property of textile goods.

Highlights
• Biosynthesized silver nanoparticles from mangosteen peel extract were spherical with particle sizes ranging from 35 to 88 nm.• A solution of 100 ppm silver nanoparticles was successfully coated on cotton fabric by the exhaustion method, with clusters of silver particles concentrated in some areas.Antibacterial properties did not significantly change after 30 washing cycles.• This silk face covering with an antibacterial layer of silver nanoparticle coated cotton fabric had 0.1 micron particle size filtration efficiency of 67.98% and airflow resistance 2.87 mm H 2 O, which met the requirement of level II ASTM performance classification.

Figure 1 .
Figure 1.Design of silk face covering.
(a).Reduction of Ag+ to AgO in the AgNO 3 reaction solutions was proposed followingAminu and Oladepo (2021).Synthesis of AgNPs in the solution was also confirmed by UVvisible spectrophotometry, which exhibited the spectrum of surface plasmon resonance (SRP) with an absorption band at 420 nm (Figure2(b)) corresponding to the classical plasmon absorption of silver metal nanoparticles in aqueous solution similar toMowafi et al. (2017) suggested that the SRP band of silver nanoparticles in solution located between 420 and 450 nm.EDS spectra recorded from the AgNPs are shown in Figure3.The broad peak at 3 keV showed the presence of silver from AgNPs reduced by G. mangostana peel extract, concurring withIbrahim et al. (2019).This peak showed the weight percentage of silver element at 73.37%.

Figure 2 .
Figure 2. The synthesis of silver nanoparticles using G. mangostana peel extract (a) Photographs showing color changes after synthesized at 90°C for 3 h (B) UV-visible spectra of the synthesized AgNPs.

Figure 3 .
Figure 3. EDS spectra recorded from a film, after formation of AgNPs different X-ray emission peaks labeled.

Figure 4 .
Figure 4. FESEM micrograph of AgNPs synthesized using the G. mangostana peel extract with accelerating voltage of 2 kV at a magnification of (A) 15000× and (B) 45000×.

Figure 6 .
Figure 6.SEM micrograph of AgNPs coated on cotton fabric with accelerating voltage of 10 kV at a magnification of 5,000× (A) CE100 and (B) CE100-30W.

Table 2 .
Viable b Value are mean of triplicate measurements.Mean values with different superscript letters are significantly different (p≤0.05).

Table 3 .
Performance properties of the barrier face covering.