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Electrospun composite nanofibre fabrics containing green reduced Ag nanoparticles as an innovative type of antimicrobial insole

In this study, polyvinyl alcohol (PVA) nanofibrous membranes containing silver nanoparticles (Ag NPs) were successfully fabricated by the combination of electrospinning and a green reduction approach. Through the electrospinning technique, uniform and smooth nanofibres can be obtained, and the Ag NPs with a narrow size distributions are well dispersed in PVA nanofibres. The investigation indicates that the mass ratio of reductant tea polyphenols and AgNO3 play a crucial role in controlling the size of the Ag NPs. More importantly, multi-layered fabrics with a layer of PVA/Ag NP nanofibrous membrane layered onto cotton substrates were developed and applied to shoe insoles. The fabricated shoe insoles with functionalized PVA nanofibres exhibit remarkable antimicrobial activity against both E. coli and S. aureus (i.e. antibacterial rate > 99%). The creation of such an encouraging fabric could establish a new optimization methodology for producing nanoengineered functional textiles.


Introduction
Textiles have long been recognized as media that provide a suitable substrate for the growth of microorganisms owing to the appropriate humidity and temperature. 1 Especially, shoe insoles are vulnerable to be contaminated by microorganisms in wearing because of the presence of the high moisture and warmth conditions, nutrients from feet sweat and greases. 2,3 The microorganisms' growth in the enclosed environment of the inner shoe may cause athlete's foot or undesired odours. Thus, it is necessary to impart an antimicrobial function into insoles to inhibit the growth of microbes. 4 Silver has been most widely recognized for its application in medical and other scientic elds, on account of its acknowledged bactericidal effect. 5 However, recent enhancements have been realized using silver nanoparticles (Ag NPs) with an attractive anti-microbial activity, which can be obtained through physical, chemical, and biological modes of reduction. [6][7][8] In particular, environmentally friendly green reductants that assist the biomimetic synthesis of Ag NPs have spurred numerous investigations. 9 The green synthesis of Ag NPs using microorganisms and plant extracts has received a great deal of attention, the latter one being the best alternative to obtain Ag NPs due to the ease and efficacy in the reduction of silver ions by the biomolecules present in plant extracts. 10 In the present study, we proposed a facile and green approach to synthesize Ag NPs, selecting tea polyphenols (TP) as the reductant. TP extracted from tea plants is a water-soluble polyphenol compound belonging to the avonoid family, mainly consisting of epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Of these, EGCG accounts for 50-60% of the total TPs. Meanwhile, such a compound exhibits highly water-soluble and excellent antioxidant properties due to its multiple pyrogallol and catechol groups. 11,12 It is also worth noting that naked Ag NPs are easily oxidized during storage and tend to aggregate due to the high surface energy, resulting in a remarkable reduction in their antibacterial activity. 13 Thus, it is of utmost necessity to maintain stability and antibacterial activity by incorporating Ag NPs with biodegradable polymers. Polyvinyl alcohol (PVA), a well-known watersoluble polymer with good biocompatibility, has been widely used in several biomedical applications, like so contact lenses, wound dressing, and tissue engineering. 14-16 Therefore, in view of the excellent characteristic of PVA, we prepared monodispersed Ag NPs which were embedded into the PVA nanobres.
Electrospinning is a sophisticated technique for the production of ultrane bres or brous membranes with diameters in the nanometre to micron range. The electrospun nanobrous membranes exhibit attractive properties, such as high specic surface area, high porosity and exibility. 17,18 Also, the nanobrous membranes can be easily functionalized through incorporating an appropriate agent or deliberately designing and controlling (manipulating) the structure of the electrospun bre. 19 More recently, there has been growing consumer demand for durable and functional apparel manufactured in a sustainable manner, implicating an opportunity for electrospun nanobrous membranes to be integrated into textile substrates. 20 For instance, in virtue of electrospinning and a doctor-blading coating modication, Xu et al. fabricated superhydrophobic polyacrylonitrile/polyurethane nanobrous membranes which contain titanium dioxide nanoparticles, with characteristics such as UV resistance, water repellency and moisture breathability. 21 Babar et al. prepared colourful cellulose acetate-based nanobrous mats with exceptional directional moisture transport performance. 22 Their ndings all indicate that electrospun nanobrous membranes integrated into textiles will be an effective and necessary strategy to provide multiple functionalities into high-value-added textiles.
Herein, a convenient method of introducing TP as the green reductant to biosynthesize Ag NPs is proposed through an in situ reduction approach in PVA aqueous solutions. Also, different crosslinking conditions for PVA/Ag NP nanobrous membranes were explored, which can guarantee a bre morphology and a brous mat pore structure aer immersing in water for a certain period of time. Meanwhile, we report a facile strategy for the fabrication of electrospun composite nanobre fabrics as an antimicrobial insole. The insole consisted of a PVA/Ag NP nanobrous membrane as an interlayer, a two-sided fusible interlining as the adhesive layer, and woven cotton as the substrate. The mechanical property and antibacterial activity of the insoles were evaluated.

Preparation of solution
The AgNO 3 aqueous solutions with TPs were prepared according to our previous work. 11 The electrospun PVA solution containing green reduced Ag NPs was prepared via three steps: rstly, the PVA aqueous solution with a concentration of 10 wt% was prepared by vigorous stirring for 2 h at 80 C. Then, the 2 wt% AgNO 3 (relative to the weight of PVA) was dissolved in the PVA solution, and the mixture was kept uninterruptedly stirred for 0.5 h when the solution temperature lowered to 65 C. Finally, 60 wt% TP (relative to the weight of AgNO 3 ) was added dropwise, and sufficient stirring for 2 h was imposed in a dark environment.

Preparation of membranes
To prepare the PVA nanobrous membrane containing Ag NPs, an electrospinning setup composed of a high voltage power supply (0-50 kV, DE-100, Ding Tong, China), a rotating drum collector, and a syringe pump (TYD 01-01, Lei Fu, China) equipped with a 10 mL syringe (with a 21 G stainless steel needle and without rubber) was used. The parameters were set as follows: a feed rate of 8 mL min À1 , a tip-to-collector distance of 15 cm, an applied voltage of 12 kV, and a roller speed of 200 rpm. The temperature and relative humidity (RH) were regulated and kept at 25 AE 2 C and 32 AE 3%, respectively. To obtain PVA/Ag NP nanobrous membrane with water stability, the samples should be crosslinked in glutaraldehyde vapor (GA) in a vacuum chamber. Also, the effects of cross-linking conditions (i.e. agent concentration, cross-linking temperature and cross-linking time) on the membrane performance were systematically investigated.

Preparation of shoe insole
The antimicrobial shoe insoles with a sandwich structure consist of ve layers, i.e., woven cotton as the substrate, twosided fusible interlining as the adhesive layer, and a PVA/Ag NP nanobrous membrane as the functional interlayer. The control specimens were prepared with a similar structure, in which the neat PVA nanobrous membrane is selected as the functional interlayer. A schematic diagram of the shoe insoles is shown in Fig. 1.

Characterization and measurements
To conrm the reducibility of TP, we measured the change of the AgNO 3 /TP aqueous solution spectra and conductivity by ultraviolet-visible spectroscopy (UV-vis, Lambda 35, Perki-nElmer, USA) and an electrical conductivity meter (DDSJ-308A, Inesa & Scientic Instruments Co., China). Fourier transform infrared spectroscopy (FT-IR) was performed using a Nicolet 5700 spectrometer (Thermo Electron Corporation, USA). Furthermore, conductivities and viscosities of polymer solutions were measured before the electrospinning. The viscosity data were obtained through a rheometer (MCR 301, Anton Paar GmbH, Austria), whose shear rate ranges from 0.1 to 1000 s À1 with a double crack coaxial cylinder (DG-26.7). The solution conductivities were achieved via a conductivity meter.
The surface morphology of the brous membranes was examined by eld emission scanning electron microscopy (FE-SEM, vltra55, Carl Zeiss, Germany) at 3.0 kV. The diameters of the nanobres were determined from the FE-SEM image using the Image Pro-Plus soware (200 bres of were randomly selected for the measurement). The distributions of Ag NPs in the nanobres were acquired via Transmission Electron Microscopy (TEM, JSM-2100, JEOL, Japan) at an accelerating voltage of 200 kV. The crystalline structure of the samples was determined by X-ray Diffraction (XRD, D8 discover, Bruker, Germany) with Cu K a radiation (l ¼ 1.5406Å) in the range of 10-90 at a scan speed of 3 min À1 .
The mechanical properties of nanobrous membranes (i.e. neat PVA and PVA/Ag NPs) were tested using a multifunctional material tensile tester (KES-G1, KATO, Japan). Specimens were cut at 5 mm width and 40 mm length and run in the instrument with a constant speed of 12 mm min À1 using standard procedures (ASTM standard 882 for thin lms and membranes). The mechanical property of the shoe insoles with neat PVA and shoe insoles with PVA/Ag NP nanobrous membrane were evaluated using a mechanical tester (Model 3344, Instron Corporation, Norwood, MA, USA) based on the ASTM standard D638. The shape of the specimens was rectangular with the size of 150 Â 25 square millimetres. A total of three experiments were performed on each sample group.
The antibacterial performances of shoe insoles against E. coli, and S. aureus were studied using a shaking ask method. A typical procedure was as follows: a mass of 0.75 g of the sample fabrics cut into small pieces of approximately 1 Â 1 square centimetres was dipped into a ask containing 50 mL of a 0.5 mM monopotassium phosphate (PBS) culture solution with a cell concentration of Â10 5 CFU mL À1 . The asks were then shaken at 150 rpm on a rotary shaker at 24 C. Given an 18 h incubation, a volume of 1 mL of microbial suspension was taken and stepwise diluted. Aer incubation at 37 C for 24 h, the number of microbial colonies plated on the nutrient on the agar plate was counted. The counts were used to calculate the surviving number of bacteria. The antibacterial rate of the specimens was calculated according to eqn (1), where W A and W N stand for the numbers of viable bacterial colonies of the shoe insoles with PVA/Ag NPs and shoe insoles with neat PVA, respectively. All the experiments were repeated three times and the results are presented as mean values.

Results and discussion
The spectra and conductivity variation of the AgNO 3 aqueous solutions with adding TP at different reaction times are displayed in Fig. 2. Fig. 2a shows the UV-vis spectra of the solutions. The peak at 435 nm in all the solutions corresponds to the surface plasmon resonance (SPR) of Ag NPs. 23 Meanwhile, the conductivity of the AgNO 3 aqueous solution increases greatly due to the existence of the H + ion which is formed in the reduction process of silver nanoparticles. Particularly, the H + ion has a higher ionic activity than the Ag + ions, 24 which is presented as the increase of solution conductivity (see Fig. 3). The variation of spectra and conductivity indicate that TP is an excellent reductant to produce Ag NPs. FTIR analysis was used to test the reducibility of TP toward Ag ions and investigate the chemical changes during the reaction process. The FTIR spectra of TP and the oxidized TP and the chemical changes before and aer oxidation are indicated in Fig. 4. The absorption band at 3346 cm À1 gets narrow and shis to 3428 cm À1 , which implies the hydroxyl ions of TP are involved in the reduction of Ag + ions, resulting in the partial destruction of the hydrogen bonds of TP and an enhancement of the attracting electron effect. Based on the comparison spectrum of oxidized TP, the emerging absorption bands at 2359 cm À1 and 1792 cm À1 in the oxidized TP was ascribed to the   stretching vibration of C]O, suggesting the formation of the orthoquinone structure in TP. 25,26 Fig . 5 shows the viscosities and conductivities of electrospun precursor solutions. The pure PVA conductivity is high since the PVA is produced from poly(vinyl esters) in alkali conditions leading to some residual ions. 27 There will be a decrease in the solution conductivity with the addition of TP, owing to increasing solution viscosity which may restrain ion migration. Besides, the PVA/Ag NP solution has the highest conductivity, consistent with the condition of the aqueous solution which can also be explained by the ionic activity theory. The solution viscosity has no noticeable change aer the addition of AgNO 3 . Nevertheless, the addition of TP or the formation of Ag NPs leads an increase of solution viscosity, suggesting that interaction between TP and PVA through hydrogen bonding may help to improve the length of the PVA molecular chains. Furthermore, the latter is more obvious than the former, which can be ascribed to TP surrounding the Ag NPs leading to more opportunity for the formation of hydrogen-bonds, as exhibited in Fig. 6.
In the electrospinning process, solution properties will be one of the key factors to determine the electrospun nanobres' morphology. 28 Fig. 7 illustrates the detailed morphology of the nanobres. Neat PVA brous membranes exhibited an even morphology feature with a randomly oriented 3D brous structure. The addition of TP resulted in a small increase in bre diameter (from 328 to 366 nm) of the resultant bres, which can be attributed to the enhanced viscosity and decreased conductivity of the solution. The average diameter of PVA/Ag NPs (i.e. 285 nm) is smaller than that of the neat PVA (i.e. 328 nm), since the conductivity of the PVA/Ag NP solution is higher than that of the pure PVA solution. Regarding the effect on the electrostatic jet, a change of conductivity plays a stronger role than changes of viscosity. The higher conductivity leads to larger residual positive charges to load on the jet surface, which directly causes a greater electrostatic repulsion, and thus bres are stretched into thinner bres with a smaller diameter.
The crystalline phase of the nanobres was determined by XRD. For the pattern of PVA/Ag NP mats, it is observed that diffraction peaks presented with 2 theta of 38.0 and 77.3 , which refer to the planes of (1 1 1) and (3 1 1) for the FCC phase Ag (JCPDS card no. 04-0783) 29 (see Fig. 8). The energy-dispersive X-ray spectroscopy (EDX), corresponding elemental mapping and high resolution TEM (HR-TEM) image further conrm the presence of Ag, and are displayed in Fig. 9. The lattice fringes can be clearly seen from the HRTEM image of Ag NPs. And the interplanar spacing is about 0.23 nm, corresponding to the Ag (1 1 1) planes. 30 In our previous reports, we found that a chelating effect between the Ag + ions and hydroxyl groups in PVA. The high viscosity of the PVA solution restricts the Ag crystal growth rate and Ag NP aggregation. 11 We also studied the effectiveness of PVA concentration and reductant dosage on the sizes of Ag NPs. 15 The PVA/Ag NP nanobres with different sizes of Ag NPs are shown in Fig. 10. With an increasing mass fraction of PVA from 10 wt% to 12 wt%, the average diameters of Ag NPs decreased slightly, which means that the viscosity has a negligible effect on size control. However, with increasing AgNO 3 , the average diameters of the Ag NPs decreased from 1.53 nm to 1.41 nm; with increasing TP, the average diameters of Ag NPs increased from 1.53 nm to 2.99 nm, suggesting a signicant effect of the reductant dosage on size control. The solution transparency declined with increasing Ag NP diameter. The     Fig. 10B inset exhibits the corresponding diluted PVA/Ag NP solutions, in which the solution with 4 wt% AgNO 3 shows the highest transparency, suggesting the smallest particle diameter. As shown in Fig. 11, the early generations of Ag atoms are just like starter seeds. The addition of TP leads to a continuous supply of Ag atoms, which add to the surface of the seeds. As a result, the nuclei will grow into nanocrystals of increasingly larger size. Besides growth via atomic addition, the nuclei and nanocrystals can directly merge into larger objects via agglomeration. This result was also conrmed by corresponding UV-vis spectra (see Fig. 10B).
In order to obtain a PVA/Ag NP brous membrane with porous structures and water stability, we used GA vapour as the cross-linking agent to treat it, and investigated the effects of cross-linking conditions. As displayed in Fig. 12, treating with high concentrations GA vapour (i.e. 50 v/v) or at a high temperature (i.e. 60 C) for 48 h, the nanobre mats swelled severely aer soaking. However, aer the treatment with GA vapour (i.e. 25 v/v) at 30 C for 24 h, the nanobre mats partially dissolved in water aer being immersed in water for 48 h, suggesting insufficient crosslinking. Furthermore, the sample aer crosslinking for 48 h became insoluble, and still maintained excellent porous bre structures even aer immersion in water for 48 h. However, the sample was excessive crosslinked aer treatment with GA vapor (i.e. 25 v/v) at 30 C for 72 h.
Mechanical properties can be considered as one of the effective factors in the application. The increase of solution conductivity leads to strong electrically driven stretching forces during the electrospinning, which will benet the orientation and stretching of the chains along the bre axis. [31][32][33] The membranes exhibited increased tensile strength from 7.31 AE 0.54 to 9.19 AE 0.83 MPa with the formation of Ag NPs, which can be caused by orientation of the macromolecule in the bres. However, the nal elongation at the breakage of PVA/Ag NP membranes decreased greatly, compared with the neat PVA brous membranes, which is shown in Fig. 13. It is indicated that due to the very high surface area and aggregation tendency of Ag NPs, the applied stress is expected to be easily transformed from the matrix onto the Ag NPs, leading to stress concentration. Also, the Ag NPs will block the movement of macromolecular chains and improve the stiffness and strength. [34][35][36] Membrane deformation behaviour images are shown in the Fig. 13 inset. What's more, the tensile stresses of the shoe insoles with a neat PVA nanobrous membrane and shoe insoles with a PVA/Ag NP nanobrous membrane show no signicant difference (see Fig. 14), suggesting the woven cotton as the principal in the insoles plays a leading role in the mechanical properties.
The antimicrobial activity was quantitatively evaluated using E. coli and S. aureus, as shown in Fig. 15. Even though the bacteria solution with a shoe insole containing neat PVA has been diluted 1000-fold, the colony count is still larger than that of the bacteria solution, with the shoe insole containing PVA/Ag NPs being diluted 10-fold. The antibacterial rates of the shoe insole with Ag NPs against both E. coli and S. aureus were greater than 99%. As we know, Ag NPs can attach to the surface of the bacteria cell membrane, disturbing the respiration functions of the bacteria cell to achieve antibacterial action. 37 Also, Ag NPs of small sizes (i.e. 1-10 nm) may serve as a vehicle to deliver Ag + more effectively to the bacteria cytoplasm and membrane. 38,39 Thus, small size Ag NPs with a larger surface area may have a higher antibacterial activity than larger ones.

Conclusions
In summary, we showed a green and facile synthesis of well dispersed small Ag NPs embedded in electrospun PVA nano-bres by applying a combination of electrospinning and a green reduction approach. Signicantly, the effect of the solution properties on the bre morphology and Ag NPs diameter were thoroughly investigated. Given the solubility of PVA, the prepared nanobrous membrane was post-treated with GA in the vapour phase, and the optimized condition was determined (i.e. GA 25 v/v, 30 C for 48 h). Furthermore, hybrid nanobre fabrics with a PVA/Ag NP nanobrous membrane layered on the substrates were developed and applied to shoe insoles and exhibited a superior antimicrobial activity, beneting from the existence of Ag NPs. Additionally, the addition of a PVA/Ag NP nanobrous membrane has no impact on the mechanical performance of shoe insoles. These properties suggest that the electrospun composite nanobre fabrics containing green reduced Ag NPs could be an appropriate candidate for antibacterial insoles. Also, this manner opens the door for diverse possibilities in the preparation of nanomaterials integrated into textiles with desired functionalities.

Conflicts of interest
There are no conicts to declare.