Synthesis and Characterization of Modified Nonwoven Fabric Samples
Samples of fabrics were modified following three step process, cleaning and activation of fabric surface, coating with silver nanaoparticles and N-halamine formation through chlorination of chitosan at fabric. The modification was carried out simply by using dip coating method. This method of coating is considered as a facile, environment friendly, requires fewer chemicals, and is cost effective. (Ceratti, Louis et al. 2015) Dip coating method is widely used in many industrial and laboratory scales methods for the coating of surface of different substrates.(Tang and Yan 2017) All experiments were carried out at room temperature. The successful attachment of modifiers on non-woven cotton fabric was investigated further using different spectroanalytical techniques. Surface Characterization of nonwoven cotton fabric modified with AgNPs, chitosan@AgNPs, and N-halamine-chitosan@AgNPs was performed by FTIR (Fourier Transform Infrared) spectroscopy, SEM, and XRD. FTIR spectra (Figure 1,a) shows characteristic peaks for AgNPs-nonwoven cotton fabric at 3348 cm-1 for O-H bonds (stretching vibration), at 2910 cm-1 for C-H bond (stretching vibration) and at 1313 cm-1 (C–H bending vibration). Peak due to the C-O bond is intense and broad, shifts to 1053 cm-1 because of AgNPs coated on nonwoven cotton fabric. These results reveal the bonding of silver nanoparticles with O-atoms of cellulose of the fabric. Coating of chitosan on the surface of AgNPs-nonwoven cotton fabric was also characterized by FTIR. Peaks at 3278 cm-1 and 2890 cm-1 belong to –OH, –NH2, and aliphatic groups and absorption peaks at 1557 cm-1 depict the N-H bending vibrations. Broad peak (at 3400 to 3500 cm-1) confirm the existence of the amine group in chitosan which is also overlapped with the broad peak of O-H. C–H bending vibration absorption at 1313 cm-1 and C-O in cellulose has an intense peak at 1053 cm-1 show the broad and red-shifted due to the bonding of AgNPs and oxygen atoms as shown in Figure 1(b). There is bond formation as N-X by halogenation of N-H groups. O-H bond (stretching vibration) in chitosan and cellulose molecules show absorption peaks at 3254 cm-1, C-H bond (stretching vibration) occurs at 2913 cm-1, and (C-H bending vibration) absorption peak occurs at 1388 cm-1. Stretching vibration of carbon and oxygen bond occurs at 1010 cm-1. Amine group in chitosan has a particular absorption peak in the region from 3400 to 3500 cm-1. Absorption peaks at 1,557 cm-1 decreased in intensity because after chlorination N-H bond converts into N-Cl bond or the formation of N-halamine occurs. FTIR absorption spectrum of N-halamine-chitosan@AgNPs-nonwoven cotton fabric is shown below in Figure 1(c).
AgNPs, chitosan, and N-halamines modified fabric surface was further characterized by SEM. SEM image of nonwoven cotton fabric is shown below in Figure 2 (a). AgNPs modified nonwoven cotton fabric show morphological changes. Some physical changes after the adsorption of silver nanoparticles on the surface of the fabric were also analyzed with SEM. It demonstrates that AgNPs coated NWFC has a smooth surface and uniform distribution of particles. SEM image of AgNPs-nonwoven cotton fabric is shown in Figure 2 (b). SEM photograph of chitosan coating on AgNPs-nonwoven cotton fabric is clear. Crystallites are uniformly distributed. Accumulation of small particles can also be observed. SEM image of chitosan/ AgNPs-nonwoven cotton fabric is shown in Figure 2(c). Loaded chlorine slightly alters the structure of chitosan coating. Aggregation of tiny particles can also be observed. SEM image of N-halamine-chitosan@AgNPs-nonwoven cotton fabric is shown-Figure 2 (d).
XRD analysis of modified and unmodified nonwoven cotton fabric was carried out to observe the structural changes. X-ray diffractometer was used for XRD analysis. Measurements were taken at 40 kV and 40 mA in 2θ range 10 to 80° with Cu-Kα radiation (λ= 0.15418 nm, 0.05 degrees step size, 1 sec per step) (Ling, Wang et al. 2019; French 2020). XRD spectra of non-woven cotton fabric are shown in (Figure 3). The peaks at 38.6o, 44.9o, 64.4o, and 77.9o obtained correspond to AgNPs-NWCF type represent the presence of silver in the fabric. (Figure 3 a) These peaks belongs to silver nanoparticles are similar as reported earlier. (Jyoti and Singh 2016). Figure 3(b) for chitosan coating on AgNPs-NWCF shows characteristic peaks at 16o and 22o reveal chitosan with crystalline (a hydrated) structure. (Jampafuang, Tongta et al. 2019). High-intensity peak at 22o represent the face-centered cubic material. X-ray diffraction pattern of chitosan@AgNPs- NWCF after formation of N-halamine shows most prominent peaks at 22o, 39o, and 46o with small less intense peaks at more diffraction angles. Figure 3(c) The small peaks represent the small size nanoparticles. (Sun and Sun 2004). All peaks are broad as compared to AgNPs-NWCF and chitosan@AgNPs-NWCF samples. It revealed that after chlorination of chitosan a new layer of chlorine atoms deposited and changes the diffraction angles. XRD pattern in addition also elucidated the nonwoven sample with and without modifiers are impurities free, surface remains the same and no additional chemical reaction takes place after coatings.
Preparation and Characterization of Antimicrobial Coating on Nonwoven Cotton Fabric
In industries, during fabric preparation, different types of chemicals or reagents are applied which protect it from insects, microbes, and other fungus attacks. Fabric surface is cleaned from residual chemicals before the surface modification process. In present study nonwoven fabric surfaces are modified using dip-coating approach. The approach is simple, eco-friendly, cost-effective, and easy to execute. Surface roughness on the nonwoven cotton fabric is achieved with a 20 mL 6M potassium hydroxide after cleaning with detergents and distilled water several times. Higher concentration of alkaline solution generated a higher number of active sites for further modification. Surface hydroxyl groups (OH) of the fabric cellulosic surface generates cellulose-OK+ on hydrolysis with potassium hydroxide. Cleaned and negatively charged surfaces of the fibers are further modified with in situ synthesis of silver nanoparticles. Alkali-treated cellulose-OK+ nonwoven fabric is dipped in AgNO3 solution. Reactive sites on the alkali-treated fabric are exchanged with Ag ions creating cellulose-OAg+. Silver ion modified surface is reduced further with ascorbic acid (C6H8O6) aqueous solution to generate silver nanoparticles (AgNPs) on the surface. Change in fabric color from white to dark brown confirms the presence and formation of silver nanoparticles at fabric. Mechanism and schematic illustrations is shown in Figure 4.N-halamine is the derivatization of chitosan polymeric materials with haloamine functional groups. This modification has attracted much attention in recent years due to its quick antibacterial response and vast potential in other biomedical and food packaging applications. Chitosan-AGNPs surfaces are positively charged that form covalent bonds with negatively charged chlorine from household bleach, requiring no additional surface modification. Therefore, chitosan-AgNPs surface is modified with an N-halamine precursor simply by dipping.
Loaded Chlorine Content Analysis
Loaded chlorine content on AgNPs- NWCF, chitosan@ AgNPs- NWCF and N-halamines-chitosan@ AgNPs-NWCF is determined with simple iodine/ thiosulphate titration procedure. 0.06 g (0.85 cm ×1.0 cm) of N-halamines-chitosan@ AgNPs-NWCF (for 30 minutes consumed 1mL sodium thiosulphate standard solution during titration. AgNPs- NWCF (Control-I), chitosan@ AgNPs- NWCF (control-II) of same weight and dimensions consumed 0 mL sodium thiosulphate standard solution. Sodium thiosulphate volume is used to calculate the loaded chlorine content on chlorinated and unchlorinated fabric, respectively as reported in earlier reports.(Liang, Chen et al. 2007; Cheng, Ma et al. 2014; Demir, Cerkez et al. 2015) N-halamines-chitosan@ AgNPs-NWCF contain 0.295% and AgNPs- NWCF, chitosan@ AgNPs- NWCF contain 0% loaded chlorine concentration. The obtained results are shown in Table S1 (supplementary information).
Antibacterial Efficacy Testing for N-halamine-chitosan@ AgNPs-NWCF
Biocidal properties of AgNPs- NWCF, chitosan@ AgNPs- NWCF, and N-halamines-chitosan@ AgNPs-NWCF samples tested against four types of bacteria as Micrococcus lutes (Gram-positive), Staphylococcus aurea (Gram-positive), Enterobacter aerogenes (Gram-negative), and E.coli (Gram-negative) following established procedures (Cheng, Ma et al. 2014) as detailed in material and methods section and schematically in Figure (5a). The fabric samples were exposed to bacterial strains at 1.7 × 105 CFU / fabric patch. Figure 5b response graph and Table S2(supplementary information) show the antibacterial results. The samples of AgNPs-NWCF and chitosan@AgNPs-NWCF chitosan offered low bactericidal activity against Micrococcus lutes, Staphylococcus aurea, Enterobacter aerogenes, and E.coli strains. The log reductions of 6.7, 6.6, 5.6, 5.4 and 7.8, 7.7, 7.5, 7.3, respectively were observed for aforementioned bacterial strains. The modified fabric samples provided about 11.2 to 11.9 log reduction within 15 min of exposure time compared to control. The inhibition efficacies of the N-halamine-chitosan@ AgNPs-NWCF improved significantly compared with other samples. The reduction in number of bacteria in media is attributed to the attachment of bacteria to the fabric samples and the inactivation by the N-Halamine and silver nanaoparticles modified fabric. It is assumed that the inactivation of bacterial growth has been achieved through transfer of positive halogen (Cl+) from N-halamine coating to the growth medium. In addition N-halamine containing stable N-X bond as shown in XRD and FTIR pattern also tends to add killing effect through contact with fabric surface.(Ahmed, Hay et al. 2009; Ren, Akdag et al. 2009; Bai, Kang et al. 2018; Wang, Huang et al. 2020) More interestingly, it is proposed that the antibacterial activity cannot be explained alone due to halogen release or contact but also combination effect of silver nanoparticles and N-halamine simultaneously. The results are similar to previous reports (Bai, Zhang et al. 2016) The N-halamine-chitosan@ AgNPs-NWCF samples inhibits bacterial growth, with Gram‐negative bacteria having lower bacterial activity than Gram‐positive bacteria. This fact is attributed to the different shapes, size, surface structures and more resistance to inactivation of bacteria over other strain. (Cheng, Ma et al. 2014) The results are equal to or consistent with the previously reports. (Liang, Chen et al. 2007; Li, Hu et al. 2013; Demir, Cerkez et al. 2015; Chylińska and Kaczmarek 2020)
Cytotoxicity studies of N-halamine-chitosan@AgNPs-NWCF
The effect of modifiers N-halamine, chitosan and silver nanoparticles on cell survivability on the Helacells was evaluated. (Figure 6) The cell viability analyses tell about the number of living cells after treatment as percentage against positive control. In the present study, cell viability % was calculated with respect to AgNPs-NWCF, chitosan@AgNPs-NWCF, N-halamine-chitosan@AgNPs-NWFC samples against sodium lauryl sulfate solution (2%) as positive control N-halamine-treated Chitosan@AgNPs-NWFC samples showed cell viability by up to 85%. These results suggested that N-halamine modified nonwoven cotton fabric not significantly inhibit the cell viability. The deviation in percentage form 100 % value may attributed to the Cl+ ions released from fabric surface and might have interfere with the cell viability measurements. Compared to N-halamine-Chitosan@AgNPs-NWFC, AgNPs-NWCF, Chitosan@AgNPs-NWCF showed 25% and 30% cell viability, respectively, which shows their significant toxicity towards HeLa cell Lines. These results suggest that N-halamine modification do not significantly possess cell toxicity; the results are in agreement with other reported results for similar studies. (Demir, Cerkez et al. 2015; Demir, Broughton et al. 2017; Grabchev, Staneva et al. 2019; Gao, Su et al. 2020) However, it is also assumed that the small toxicity observed in the present experiment may due to release or dissociation of Cl+ into cell medium with time from N-halamine modified chitosan@AgNPs-NWFC. The release can be controlled by optimizing the chlorine contents at nonwoven cotton fabric and with repeated washing with water without affecting fabricated fabric properties.
Photostability and Shelf Life Stability
Nonwoven cotton fabric samples are investigated for Photostability and shelf life at room temperature. It has been reported in the literature that N–Cl bond in N- halamine shows sensitivity towards light irradiations. The dissociation of bonds increased with exposure to UVA light. (Ma, Li et al. 2019). The shelf life (storage) stability of bound chlorine in N-halamines-chitosan@AgNPs-nonwoven fabric under UVA lamp and dark environment for 12 weeks are shown in Figure S1 ( Supplementary Information). N-halamines-chitosan@AgNPs-NWCF stored in dark environmental conditions retained most of their initial chlorine loadings for 6 weeks. N-halamines-chitosan@AgNPs-NWCF lost only 20% of chlorine after 12 weeks of storage in darkness. It is observed that N-halamines-chitosan@AgNPs-NWCF excellent storage stability and retained 80%± 10 of their chlorine content after 12 weeks in a dark environment. However, N-halamines-chitosan@AgNPs-NWCF is somewhat less stable under the light. It is noted that the N–Cl bond dissociation increased with exposure to UVA light. When stored under light, N-halamines-chitosan@AgNPs-NWCF samples lost only 70% of the oxidative chlorine over 12 weeks of storage. Photostability of N-halamine modified data is comparable with previous reports. (Chylińska and Kaczmarek 2020) It can be concluded that the present modification method is stable, simple, low cost and environment friendly yet to be established in laboratories for various biomedical applications.