New Textile for Personal Protective Equipment—Plasma Chitosan/Silver Nanoparticles Nylon Fabric

: Fabric structures are prone to contamination with microorganisms, as their morphology and ability to retain moisture creates a proper environment for their growth. In this work, a novel, easily processed and cheap coating for a nylon fabric with antimicrobial characteristics was developed. After plasma treatment, made to render the fabric surface more reactive sites, the fabric was impregnated with chitosan and silver nanoparticles by simply dipping it into a mixture of different concentrations of both components. Silver nanoparticles were previously synthesized using the Lee–Meisel method, and their successful obtention was proven by UV–Vis, showing the presence of the surface plasmon resonance band at 410 nm. Nanoparticles with 25 nm average diameter observed by STEM were stable, mainly in the presence of chitosan, which acted as a surfactant for silver nanoparticles, avoiding their aggregation. The impregnated fabric possessed bactericidal activity higher for Gram-positive Staphylococcus aureus than for Gram-negative Pseudomonas aeruginosa bacteria for all combinations. The percentage of live S. aureus and P. aeruginosa CFU was reduced to less than 20% and 60%, respectively, when exposed to each of the coating combinations. The effect was more pronounced when both chitosan and silver were present in the coating, suggesting an effective synergy between these components. After a washing process, the antimicrobial effect was highly reduced, suggesting that the coating is unstable after washing, being almost completely removed from the fabric. Nevertheless, the new-coated fabric can be successfully used in single-use face masks. To our knowledge, the coating of nylon fabrics intended for face-mask material with both agents has never been reported.


Introduction
Fabrics have an ideal environment for the growth of microorganisms. They are porous, retain water and, as they are close to human body, their normal temperature is approximately of 37 ºC. This fact is even more stressful taking in consideration the pandemic state that we are living in. The constant use of facial masks and protective clothes, if not used properly, increases the risk of infection. A fabric with antimicrobial properties is essential to overcome this problem.
The antimicrobial properties of silver are known for many years. Silver is currently used in several medical devices like wound dressings, coating of surgical instruments and prostheses [1]. With the advances of technology, silver nanoparticles have been widely prepared and demonstrated to possess high antimicrobial potential. They now considered a good alternative to antibiotics, although its use for medical applications can be hindered due to its high toxicity to the patient and the environment [2]. It is known that silver particles can enter the human body by different ways and accumulate in different organs, but most important it can cross the blood brain barrier reaching the brain [1]. So, there is the need to decrease the silver nanoparticles toxicity but keeping its antimicrobial properties. One approach is the incorporation of the silver particles in a biocompatible polymer like chitosan, which has intrinsic anti-microbial properties, creating a synergy between the two materials [3,4].
An interesting fact is that silver nanoparticles have demonstrated an efficient inhibitory activity against human immunodeficiency virus (HIV-1) [5,6] hepatitis B virus (HBV) [7], respiratory syncytial virus herpes simple's virus type 1 [8,9] monkeypox virus plaque formation [10] as well as influenza H1N1 virus [11]. A very recent study claims that silver maybe effective on the treatment and prevention of COVID -19 and SARS-COV-2 [12].
So, on this work a nylon fabric was functionalized by a simple dip coating method with a mixture of different concentrations of silver nanoparticles and chitosan. The characteristics of the textile were analysed, as well as the synergistic effect of the active agents. Silverbased nylon fabric has long been known to act as antimicrobial agent for wound therapy helping on the mitigation of wound infection and inflammation [13]. The synergistic effect of silver and chitosan has also been proven to be effective for preventing the agglomeration of silver nanoparticles and improve its biocompatibility. To our knowledge a nylon coating intended for face masks material with both agents has never been reported.

Materials
Chitosan (ChitoClear hq95-43000) was purchased from Primex (Iceland) and polyamide taffeta with 52 warp and 32 weft yarns, areal density 100 g/m² from Lemar (Guimaraes, Portual). All the other materials were purchased from Sigma-Aldrich and used without further purification.

Nanoparticles Synthesis
A 0.02 gL -1 colloidal dispersion of silver nanoparticles (AgNPs) doi: 10.37532/jftte.2020.8 (5).200 was prepared using the modified stepwise method of the conventional reduction technique described by Lee and Meisel [14]. All solutions were prepared in distilled water. During the process the dispersion was mixed vigorously. A volume of 10 ml of 1% trisodium citrate (Na 3 C 6 H 5 O 7 ) was added drop-by-drop to 100 ml of 1 mM silver nitrate (AgNO 3 ) previous heated to boiling point in a 250 mL flask reaching a final concentration of 3.8 mM final concentration. The pH of the reaction was adjusted to 7.7 by adding HNO 3 or NaOH. The solution was heated again to boiling temperature until colour's change was evident (pale yellow). At this stage the solution was cooled to room temperature while mixing.

Preparation of Chitosan solution
Chitosan was prepared in a concentration of 0.5 wt %. Chitosan (0.5 g) was dissolved in 2 mL of acetic acid (glacial) to completely dissolve the chitosan and distilled water was further added until reach 100 ml of solution and stirred at 300 rpm for 30 min.

Nylon fabric plasma treatment
To improve the adhesion of the coating, plasma treatment was applied previous to the application of the antimicrobial agents. The DBD plasma treatment was performed conducted in a semi-industrial prototype machine (Softal Electronics GmbH/University of Minho, Guimarães, Portugal) working at room temperature and atmospheric pressure, using a system of metal electrode coated with ceramic and counter electrodes coated with silicon with 50 cm effective width, gap distance fixed at 3 mm and producing the discharge at high voltage 10 kV and low frequency 40 kHz. The discharge power supplied by the electrodes and the speed may vary, with maximum discharge of 1.5 kW and speed of 60 m min −1 . The machine was operated at the optimized parameters: 1 kW of power, velocity of 4 m min −1 , 5 passages corresponding to a dosage of 2.5 kWmin m −2 as previously reported [15].

Functionalization of nylon fabric with antimicrobial agents
Nylon fabrics were coated with chitosan, silver NPs and a mixture of both using different concentration of mixed solutions as described in Figure 1. Dilutions were made starting with the mother solution of chitosan (CH) and silver nanoparticles (Ag) with the following ratio CH/Ag: 1:0; 0:1; 6:4; 7:3; 8:2; 9:1 ( Figure 1A). The coating solution was applied to the polyamide fabric by a simple dip coating technique. The fabric was dipped in each solution for 5 minutes at room temperature ( Figure 1B). The coated material was then placed in an oven at 50°C for 20 minutes.

Scanning Electron Microscopic (SEM) Analysis
The morphological characterization of Ag NPs and the combination of both Ag NPs with chitosan were visualized by SEM, which was also used to assess the mean particle average of the obtained NPs. Further, the functionalized nylon fabrics with both antimicrobial agents before and after application of plasma treatment was also visualized with SEM. An ultra-high-resolution Field Emission Gun Scanning Electron Microscopy (FEG-SEM), NOVA 200 Nano SEM, FEI Company was used. Secondary electron and backscattering electron images were performed with an acceleration voltage of 5kV and 15kV, respectively. Samples were previously coated with an Au-Pd film (80-20 weight %) in a high-resolution sputter coater, 208HR Cressington Company, coupled to a MTM-20 Cressington High Resolution Thickness Controller.

Reflectance
UV-visible absorption intensity employing diffuse reflectance spectrophotometer was used to monitor the changes associated with modification of nylon fabric with silver nanoparticles. The UV-visible spectra for all the samples were recorded in the wavelength range of 200-800 nm at room temperature and the absorbance of the peak at 410 nm, characteristic of silver surface plasmon resonance (SPR).

Antimicrobial assays
The antimicrobial activity was determined using the standard shake flask method (ASTM-E2149-01) with some modifications. S. aureus (ATCC25923) and P. aeruginosa (PAO1) inoculum was prepared using a single colony from the corresponding bacterial. The culture was grown overnight in sterile nutrient broth (NB, Sharlab, Spain) at 37 o C, 230 rpm. The material was incubated with 5 mL of bacterial suspension (3E8 cell/mL in NaCl 0.9% pH 6.5) at 37 o C and 100 rpm, for 2 hours. The withdrawn suspensions the bacteria viability was determined by flow cytometry as described below.
A volume of 2.5 µL and 10 µL of the SYTO-BC (50 µM stock solution) mixed with 5µL of PI (1.5 mM stock solution) was added to 250 µL of bacterial suspension of S. aureus and P. aeruginosa, respectively. Samples were analysed after at least 15 min of incubation at room temperature. Cell debris and other particles were excluded by gating as unstained particles.
Setting parameters and data analysis: Live/dead bacteria was assessed using a EC800 Flow Cytometry Analyzer (Sony Biotechnology Inc., Champaign, IL, USA). SYTO-BC and PI were excited by a diode blue laser (488 nm); The green fluorescence emitted by bacteria with intact membrane, here designed as "live", was detected using a 530/50 nm band pass and the red fluorescence emitted by bacteria with damaged membrane, here designed as "dead", was detected using a 615/30 nm band pass filter. Therefore, for convenience of the data analysis, double stained bacteria corresponding to "compromised bacteria" were considered as "dead". Fluorescence signals were amplified with the logarithmic mode. A sample volume of 100 µL was analysed at a flow rate of 10 µl/min. Total events of 40000 cells were counted. Every sample was run in triplicate for reproducibility of the experiment. Data analysis was performed on the EC800 software version 1.3.6. (Sony Biotechnology Inc., Champaign, IL, USA).  increase of the NPs incorporation on the material surface mainly on the material previously treated with plasma. A significant decrease was observed when the highest concentration of AgNPs was incorporated indicating that the improve adhesion in presence of plasma is driven by a concentration related mechanism (Figure 4).
The antimicrobial property of the nylon fabric (PA) was further assessed taking into account different combinations of chitosan (CH) and silver nanoparticles (Ag). Overall, different antimicrobial responses were obtained for a gram-positive bacterium (S. aureus) and for a gram-negative bacterium (P. aeruginosa) ( Figure 5). The bactericidal activity was higher for the S. aureus than for P. aeruginosa in all the combinations of chitosan and silver nanoparticles. The percentage of S. aureus CFU was reduced to less than 20% when exposed to each of the combinations of chitosan and silver nanoparticles, the fabric with chitosan and silver nanoparticles 70/30 was the most effective. P. aeruginosa was less susceptible to the antimicrobial treatment. Even the treatment with chitosan and silver nanoparticles 0/100 was ineffective. The combination of chitosan with silver nanoparticles resulted in 60% reduction of P. aeruginosa, approximately.

Results and Discussions
In this work, the synthesis of AgNPs was performed using a wellknown chemical procedure described by Lee and Meisel [14], which usually results in NPs colloidal solution that is cytotoxic in contact with mammalian cells [16,17]. The combination of AgNPs on a polymeric matrix results in the improvement of the biocompatibility without jeopardizing the antimicrobial effect of the NPs [18,19]. That is why a nanoformulation containing chitosan solution with dispersed AgNPs to further apply on the nylon fabrics was developed. The chitosan solution was found to act as a stabilizer and dispersant of the AgNPs. As depicted in Figure 2, the AgNPs agglomerate when they are in an aqueous colloidal solution (Figure 2A), while in the presence of chitosan they are well dispersed, keeping an average size of 24.8 ± 4.1 nm (Figure 2B), thus acting as a surfactant for the stabilization of the NPs. This is very important in terms of biomedical applications. Chitosan besides acting as a surfactant, also possess important antimicrobial properties. The charged amino group present in its chemical structure confers a biodegradable and biocompatible character to the mixture [18].
The AgNPs, chitosan, and the mixture of both were impregnated in the nylon fabrics through a dip coating method on the material with and without a previous plasma treatment. Figure 3 shows the surface of the textile material.
It could be observed that the plasma treated nylon control has a smoother surface, which is the result of a surface etching induced by the application of plasma, whose rate depends on the degree of crystallinity of the material [20]. Overall, the application of all types of antimicrobial agents (AgNPs, chitosan and the combination of both) is clearly influenced by the plasma treatment. The plasma treated material has a higher amount of coating material on its surface, although it is not fully covered by any of the materials. The layers are more prominent on plasma treated samples (Figure 3, right column). As expected, when the AgNPs are applied without the chitosan they agglomerate in one part of the fibre while when applied together with the chitosan a layer of this combined coating is clearly observed (Figure 3, last line).
The nylon fabric was further tested for incorporation of increased concentration of silver on the chitosan-based nanoformulations. Reflectance measurements were performed to qualitatively assess the amount of silver immobilized on the material surface. The reflectance was measured at the higher observed peak for Ag NPs (410 nm) assigned to the silver surface plasmon resonance usually located in between 405 nm -425 nm [21][22][23]. The lower the reflectance the higher is the amount of Ag in the material surface. Thus, the decrease observed in Figure 4 on the reflectance at 410 nm, indicates an doi: 10.37532/jftte.2020.8(5).200 In order to evaluate the possibility of this material to be reused, the fabric was subjected to a washing treatment, after which the percentage of live bacteria increased significantly (p<0.001) for all the combinations of chitosan and silver nanoparticles. This result indicates that the chitosan and silver nanoparticles coating is very unstable, being almost completely removed from the fabrics as shown Figure 5. The two bacteria tested, S. aureus and P. aeruginosa, are among the most common infection agents and representative of each group (gram positive and gram-negative bacteria, respectively). As these bacteria are often found in skin and upper respiratory tract, they will pose a serious problem in the use of facial masks, as fabric is in contact with skin for many hours. The infections caused by these two infectious agents can be minor to life threatening, being particularly dangerous for immunocompromised patients [24][25][26].
P. aeruginosa is a gram-negative bacillus characterized by a cytoplasmatic membrane with a symmetric phospholipid bilayer and asymmetric outer membrane with a phospholipid inner face and a lipopolysaccharide outer layer forming a permeability barrier [27,28].
In the presence of silver nanoparticles, the first event is the adsorption of silver nanoparticles into the surface of the cell membrane. This will undermine the membrane integrity, inducing the particles transport into the cytoplasm influencing several metabolic processes. It has been reported that silver nanoparticles influence the equilibrium of the oxidation and anti-oxidation mechanism leading to an excess of reactive oxygen species, can cause DNA aggregation and protein degradation leading to the cell death [29][30][31]. This process described by Liao et al it can take up to several hours, in our case the bacterium was only in contact with the nanoparticles for 2h, which may indicate that was not enough contact time to obtain a higher death rate.
An important difference between gram-negative and grampositive bacteria is the composition of the cell wall, which may justify the distinct effects observed on the presence of the silver nanoparticles [32].
A gram-positive bacterium does not have an outer membrane, similar to the gram-negative bacteria, but has a thick layer of peptidoglycan complemented by anionic glycopolymers know as teichoic acids [33]. This structure is known to offer less resistance to the passage of some substances, like antibiotics and it has been described that silver can perforate the peptidoglycan cell wall [34,35].

Conclusion
The developed coating demonstrated to be highly effective against the most common gram-positive (S. aureus) and gramnegative (P. aeruginosa) bacteria. Even though the coating seems to be unstable after a washing treatment, this can be applied in fabric intended to facial masks, as it will prevent infections. According to the literature the presence of this silver may also prevent an infection by virus in particular by SARS-COV-2. To our knowledge the coating of nylon fabrics intended for face masks material with both agents has never been reported. The new-coated fabric can be successfully used for a single use face mask.