Journal of Fashion Technology & Textile EngineeringISSN: 2329-9568

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Research Article, J Fashion Technol Textile Eng Vol: 4 Issue: 2

Silver-loaded Antibacterial Alginate Nanofibres: Preparation and Characterization

Forhad Hossain M1* and Hugh Gong R2
1Department of Wet Process Engineering, Bangladesh University of Textiles, Dhaka, Bangladesh
2School of Materials, Manchester University, United Kingdom
Corresponding author : Forhad Hossain M
Assistant Professor and Head, Department of Wet Process Engineering, University of Textiles, Dhaka, Bangladesh
Tel: +8801714322070
E-mail: [email protected]
Received: May 23, 2016 Accepted: June 21, 2016 Published: June 26, 2016
Citation: Forhad Hossain M, Hugh Gong R (2016) Silver-loaded Antibacterial Alginate Nanofibres: Preparation and Characterization. J Fashion Technol Textile Eng 4:2. doi:10.4172/2329-9568.1000137

Abstract

Sodium alginate is a very popular thickening agent used in food, pharmaceutical and textile industry. It is also used in different biomedical applications and wound dressings due to its biocompatible properties. In this study, however, this biopolymer was electro spun from aqueous solution by combining a small portion of polyethylene oxide (PEO) as carrying polymer. In the spinning solution, 70:30 Na-alginate/PEO of total 4.0 wt. % was used to obtain bead-free nanofibres from electrospinning. To provide insolubility and antibacterial properties, these fibres were then chemically modified by treating with CaCl2 and AgNO3 in ethanol absolute solution. During chemical treatment process, 1.0 and 5.0 wt. % of CaCl2, and 0.5 and 1.0 wt. % of AgNO3 were used. The nanofibres structure and morphology were investigated by Field Gun Emission Scanning Electron Microscope (SEM), Energy Dispersion X-ray (EDX), and Fourier Transform Infrared Spectroscopy (FTIR). The results prove that silver-loaded antibacterial alginate nanofibres have been successfully produced.

Keywords: Anti-bacterial; Electrospinning; Nanofibres; Poly (ethylene oxide); Sodium alginate

Keywords

Anti-bacterial; Electrospinning; Nanofibres; Poly (ethylene oxide); Sodium alginate

Introduction

Alginate is a biodegradable and biocompatible renewable material available in nature, traditionally used as thickening agent in printing industry, additives in food industry, and formulation of medicine in pharmaceutical industry [1,2]. It can be fabricated as a nanofibrous mats by electrospinning technique for biomedical and healthcare applications, including tissue engineering, drug delivery, artificial body organs, and wound dressing materials [2-4]. However, sodium alginate is not suitable for electrospinning. This is because Na-alginate is a polyelectrolyte polymer which contains polyanions like chitosan [5]. The polyanions of Na-alginate molecule repulse each other and hinder molecular chain entanglement. Moreover, Na-alginate especially with high guluronic acid (G) content, which may turn into a gel easily, becomes too viscous for electrospinning at slightly higher concentration. These problems can be overcome by adding a suitable copolymer and a small amount of surfactant to the Na-alginate solution. Mincheva et al. found that the hydrogen bonds between the polyelectrolytes and non-ionic water-soluble copolymer poly ethylene oxide (PEO) favour electrospinning [6]. Addition of a small amount of surfactant along with PEO reduces the surface tension and viscosity to improve the electrospinnability of the spinning solution which allows increasing the alginate content in the solution [7,8]. As a result, the spinnability and fibre formation of alginate/PEO would increase further, even at higher alginate content in the solution.
The as-spun Na-alginate/PEO are water soluble and they are unable to sustain in aqueous environments which might present in the targeted biomedical applications. Thus, these fibres need to be converted to insoluble form to attain the structural benefits of nanofibrous mats. Moreover, the most widely used antibacterial agent, silver can be incorporated with the alginate nanofibres to gain antibacterial properties of nanofibres. These silver-containing nanofibres can be used in precise biomedical applications and wound care products. Wiegand et al. studied the effectiveness of wound dressings made from alginate and silver containing alginate. They found that alginate shows antibacterial activity with considerable effectiveness to reduce elastase, proinflammatory cytokins, and free radical formation. However, the alginate dressings containing silver showed improved effectiveness against these wound parameters with higher antibacterial activity [9]. Some studies [10-12] revealed silver loaded electrospun nanofibres from different biopolymers. Most of them used silver nanoparticles in the spinning solution. However, in this study, the silver particles have been incorporated in the fibres by post-treatment of the as-spun Na-alginate/PEO nanofibres with AgNO3 in ethanol absolute solution.
Chemical treatment is normally used to alter the existing properties of materials to achieve certain functional properties such as strength, stability, water resistance, fire resistance, antibacterial activities, etc. Several studies [8,13,14] have been carried out to convert as-spun Na-alginate nanofibres to insoluble Ca-alginate nanofibres. These researchers made Ca-alginate nanofibres by treating Naalginate nanofibres with CaCl2 through a certain chemical treatment process. Le et al., demosntrated a chemical modification process to produce Ag-, Na-and Ca- alginates fibres in wet spinning process. In that process, the extruded filaments of Na-alginate were collected in a bath containing calcium chloride and silver nitrate mixed solution. At this stage an ion exchange reaction converts the sodium alginate fibres to insoluble antibacterial Ag-, Na-, and Ca- alginates fibres [15]. It is easy to incorporate silver ions onto the calcium alginate fibres by treating with silver nitrate solution [1]. However, due to pre-reaction of silver nitrate and calcium chloride in the solution at the mixing stage in ethanol absolute, in this study, the Na-alginate/ PEO nanofibres was treated with calcium chloride and silver nitrate solution consecutively. The following reactions take place during this wet chemical treatment process:
2Na-alginate + CaCl2←→Ca(alginate)2 + 2NaCl
Na-alginate + AgNO3←→Ag-alginate + NaNO3
Experimental Procedures

Materials

Na-alginate (high G) powder was supplied by ConvaTec UK, PEO powder (Mw=900,000) and Triton X100 were purchased from Sigma-Aldrich. The syringe (10 ml, BD Plastipak) and needle (18G) were supplied by Beckton Dickinson Plastipak. Silver nitrate (AgNO3) (99.9+%, metal basis) granules were purchased from Alfa Aesar, A Johnson Mathew Company. Calcium chloride (CaCl2, minimum 90%) powder and ethanol absolute were purchased from BDH Laboratory UK and Fisher Scientific UK, respectively. All the chemicals were used without further modification or purification.
Preparation of spinning solution: Predetermined amount of Naalginate powder and PEO were added with distilled water in a 60 ml bottle by using a precision electronic balance to prepare 2-6 wt. % of 50:50-90:10 Na-alginate/PEO solution. To improve the homogeneity of the spinning solution 0.5 wt. % Triton X100 surfactant was also added to the solution. Then the sealed bottle was transferred to a magnetic stirrer and left for overnight at room temperature (around 20°C) to obtain a uniform spinning solution.
Preparation of treatment solution: Predetermined amount of CaCl2 powder (1.0 g) was dissolved in 99.0 g ethanol absolute in a bottle by using a precision electronic balance to prepare 1 wt. % CaCl2 solution. Then the sealed bottle was transferred to a magnetic stirrer and the solution was gently stirred at room temperature (around 20°C) for overnight to obtain a uniform solution. Similarly, 5.0 wt. % CaCl2, 0.5 wt. % and 1.0 wt.% AgNO3 solutions were prepared for carrying out the chemical treatment processes.

Methods

Instrumental setup and electrospinning process: A horizontal electrospinning device was used to make Na-alginate/PEO nanofibres. A syringe, loaded with spinning solution was set in the pump. Electrospinning trials were carried out using 2-6 wt.% polymer concentration and 50:50-90:10 blend ratios. The process parameters were adjusted to 12-20 cm working distance, 1.0-0.3 ml/h feed rate, and 9-12 kV applied voltage to obtain uniform fibres. The uniform fibres were found using 4 wt.% polymer concentration with 70:30 blend ratio at 16 cm working distance, 0.4 ml/h feed rate, and 10.5 kV electric potential. The electrospun fibres were collected on an aluminium foil. After allowing 1h of electrospinning, the foil with deposited fibres was collected for drying 24 hours at room temperature to remove the residual solvents. All of the electrospinning experiments were carried out at room temperature.
Chemical treatment: A total of 7 petri-dishes were prepared in a fume cupboard to perform this experiment. As-spun Na-alginate/ PEO nanofibres were placed in a petri-dish containing ethanol absolute (about 25 ml) to soak the sample. The petri-dish was shaken gently for 10 minutes to dissolve the PEO of the nanofibres. The sample was then transferred in a petri-dish containing 1.0 wt. % CaCl2 solution in ethanol absolute and stood for 10 minutes. At this stage, an ion exchange reaction would take place between sodium ion (Na+) of Na-alginate and calcium ion (Ca2+) of CaCl2. As a result, the Naalginate nanofibres would be converted into Ca-alginate nanofibres. The sample was then transferred to another petri-dish containing 0.5 wt. %AgNO3 solution, and stood for 10 minutes. At this stage, the Ca-alginate nanofibres would be converted into Ag-Ca-alginate nanofibres. Then, the sample was washed in ethanol absolute solution to remove precipitated and unreacted salts. Finally, the sample was dried at room temperature (around 20°C).This process is repeated to prepare a total of 4 samples, treated with different concentrations of CaCl2 and AgNO3, according to Table 1. All of these experiments were carried out at room temperature.
Table 1: Sample plan and treatments conditions.
Morphology and characterization: The morphology and characterization of nanofibre mats were investigated by Field Emission Gun Scanning Electron Microscope (SEM) and Energy Dispersion X-ray (EDX) (PHILIPS XL30 FEG-SEM), and Fourier Transform Infrared Spectroscopy (FTIR) (Model: NICOLET5700 FT-IR; Manufacturer: Thermo Electron Corporation). For SEMEDX study, the samples of 0.5 cm×0.5 cm in size were adhered on a specimen stub by carbon tape specified for SEM purpose. Then the samples were coated with carbon using gatan Precision Etching Coating System (model: 682). The SEM images were taken at 2000×, 10,000× and 20,000× magnifications. The SEM operating parameters were set at 6kV accelerating voltage and spot size 3. The fibre diameters were manually measured using the line-drawing feature in ImageJ [16] software from 50 randomly selected fibres in the 10,000× and 20,000× magnification images at 3 different focal points. The ImageJ line drawing feature reports the line length in pixels; and pixels are converted to standard units of length measurements using the SEM image scale bar. However, for EDX analysis, the scanning was done at 2000× magnification and the parameters were set at 10kV accelerating voltage and spot size 3.
The chemical structure of Na-alginate/PEO and chemically modified alginate fibres were analysed by FTIR. It can produce an infrared spectrum of absorption, emission, photoconductivity or Raman scattering of a material. The infrared spectra of absorption mode of the nanofibres are obtained from FTIR spectrometer which is connected with a PC. The spectra were scanned with a spectral range of 4000-400 cm-1 with 32 scans and a resolution of 4 cm-1.
The solubility behaviour of the alginate nanofibres was tested in accordance with BS EN 13726-1:2002 section 3.7 dispersion and solubility of hydrogel dressings. The samples (2 cm × 2 cm) were put in 20 ml of water in a petri-dish and left in an incubator at 37°C for 24 hours. Visual assessment was done at laboratory atmospheric conditions to examine the solubility of nanofibres.

Results and Discussion

Morphology and characterization of nanofibres
The chemical treatments of the Na-alginate/PEO nanofibres were carried out with CaCl2 and AgNO3 in ethanol solution. The effect of chemical treatment process on morphology was observed from SEM images. Figure 1[A1][B1][C1][D1][E1] show the SEM images of nanofibres of different state and Figure 1[A2][B2][C2][D2][E2] show the corresponding fibre size distribution. It is observed that the 4 wt.% of 70:30 of Na-aloginate/PEO solution with 0.5 wt.% Triton X100 surfactant yielded smooth fibres having an average diameter of 141nm with very few thick or spindle-like fibres. The morphology of as-spun Na-aloginate/PEO fibres (Figure 1[A1]) is significantly changed in the chemically treated fibres (Figure 1[B1] [C1][D1][E1]). The uniformity of the nanofibres deteriorates in some extent due to chemical treatment process – thick and thin places appear in the fibres. It is observed that the sample T0.5/1.0Ag/Ca and T1.0/1.0Ag/Ca have retained the structural consistency of the as-spun nanofibres. However, the samples T0.5/5.0Ag/Ca and T1.0/5.0Ag/Ca, treated with higher concentration (5.0 wt. %) of CaCl2, have many beads with broken fibre structure. It can be assumed that the higher concentration of CaCl2 adversely affects the nanofibrous structure whereas the concentration of AgNO3 has little effect on the fibres.
Figure 1: SEM images ×10K and fibres size distribution of as-spun 70:30 Na-alginate/PEO nanofibres [A1][A2]; T0.5/1.0Ag/Ca [B1][B2]; T1.0/1.0Ag/ Ca [C1][C2]; T0.5/5.0Ag/Ca [D1][D2] and T1.0/5.0Ag/Ca [E1][E2].
Figure 2 shows the average diameter of the as-spun Na-alginate/ PEO nanofibres, T0.5/1.0Ag/Ca, T1.0/1.0Ag/Ca, T0.5/5.0Ag/Ca and T1.0/5.0Ag/Ca that found to be 141, 131, 130, 136 and 134 nm, respectively. These results indicate that the average diameter of the fibres was decreased due to chemical treatment. This may have happened due to the removal of PEO from the fibres. As shown in Figure 1[A2] [B2][C2][D2][E2], the fibre size distribution becomes wider with the concentration of CaCl2 and AgNO3 in chemical treatment process. This also indicates that the concentration of CaCl2 have negative effect on the uniformity of the fibres. However, the concentration of CaCl2 may increase the diameter of the fibres.
Figure 2: Average diameter of the as-spun Na-alginate/PEO and chemically treated fibres.
It is also observed that a large number of spherical particles adhere to the fibres in the samples. It is assumed that the spherical particles are generated from AgNO3, which represents the presence of silver (Ag). As shown in Figure 1[B1], at lower concentrations of AgNO3 and CaCl2, the silver particles are uniformly distributed over the fibres. However, an aggregation of the silver particles is observed when the concentration of AgNO3 is increased from 0.5 wt. % to 1 wt.% at the same 1 wt.% CaCl2 concentration (Figure 1[C1]). The sizes of the silver particles in the fibres were manually measured using line-drawing feature in ImageJ [16] software from 50 randomly selected particles in the 10,000× and 20,000× magnification images of the chemically treated samples. Figure 3 presents the size distribution of the silver particles. The SEM images, as shown in Figure 1[B1] [C1][D1][E1], show that the silver particles are distributed over the fibres uniformly although an aggregation is observed in the sample T1.0/1.0Ag/Ca. The average diameter of the silver particles is found to be 210 nm with good size distribution.
Figure 3: Size distribution of the silver particles in the chemically treated alginate nanofibres.
EDX analysis
Energy Dispersion X-ray (EDX) analysis was carried out to examine the presence of the expected elements. EDX measures the key elements such as silver (Ag), sodium (Na), calcium (Ca), oxygen (O) and chlorine (Cl) present in the nanofibres. Table 2 shows the average amount of these individual elements and Figure 3 shows the relative amount of these elements present in the nanofibrous mats. The amount of the silver is higher in all the samples compared to other elements (i.e. Na, Ca, Cl, and O). The results reveal that the concentration of AgNO3 has only minor effect on the silver content at constant CaCl2 concentration, but the relative silver content increases significantly when the concentration of CaCl2 decreases from 5.0 wt.% to 1.0 wt.% at constant AgNO3 concentration. However, the calcium content increases significantly with increasing CaCl2 from 1.0 wt.% to 5.0 wt.% at constant silver concentration. Figure 4 shows that the relative amounts of silver, calcium and sodium present in the fibres are consistent with each other though the sample with higher content of calcium showed little inconsistency. From these results, it is assumed that the Ag-Na-Ca-alginate antibacterial nanofibres are successfully produced.
Table 2: The amount of different elements present in the dressing samples.
Figure 4: Comparison of the chemically treated samples on basis of composing elements.
FTIR analysis
Fourier Transform Infrared Spectroscopy (FTIR) was used to observe the spectral peak variation between the Na-alginate/PEO and post-treatment alginate nanofibres. Figure 5 [I,II] show the FTIR spectra of as-spun Na-alginate/PEO fibres and chemically treated alginate fibres within 4000-500 cm-1 and 3400-2000 cm-1 spectral range. Some essential interactions at 1100-1090 cm-1, 1650-1550 cm-1, and 3300-3400 cm-1 were observed between Na-alginate and PEO in as-spun Na-alginate/PEO nanofibres that also found in other studies [14,17,18]. These interactions reduce the repulsive force between polyanionic sodium alginate molecules, which ultimately assists electrospinning.
Figure 5: The FTIR spectra of as-spun Na-alginate/PEO nanofibres [A], T0.5/1.0Ag/Ca [B], T0.5/5.0Ag/Ca [C], T1.0/1.0Ag/Ca [D] and T1.0/5.0Ag/Ca [E].
The spectral peaks between the Na-alginate/PEO fibres and chemically treated fibre are changed significantly because of the chemical treatment. It is observed that the peak at 2883 cm-1 in the Na-alginate/PEO nanofibres represents the –CH2 group which is diminished in the chemically treated samples. This indicates that the PEO in the chemically treated fibres is removed completely. The peaks 3346 – 3336 cm-1 include hydrogen bonds which represent the –OH group. The peak for –OH group that occurs at 3346 cm-1 for Na-alginate/PEO, is moved to 3336, 3340, 3340 and 3336 cm-1 for T0.5/1.0Ag/Ca, T0.5/5.0Ag/Ca, T1.0/1.0Ag/Ca and T1.0/5.0Ag/ Ca respectively. Thus, the frequency of –OH group of Na-alginate/ PEO is shifted to lower bands due to the chemical treatment. This indicates that the Na-alginate nanofibres are turned into calcium alginate nanofibres.
Solubility of nanofibres
Chemical treatment was carried out to alter the solubility behaviour of Na-alginate nanofibres as well as to incorporate silver antimicrobial into the fibres. Insolubility of the nanofibres is important to ensure the functional benefits from nanofibrous structure fully. Retention of fibrous structure in aqueous environment is the most important parameter in biomedical applications. For instant, it is believed that the porous structure of the nanofibrous dressing promotes the growth and spread of epithelium cells in the wounds [19,20]. However, if the nanofibres of the dressings dissolve in contact with wound fluid they will not be able to perform their functions. Thus, it is expected that the alginate fibres developed in the current study will retain their nanofibrous structure when they come in contact with biological environment. Therefore, the solubility behaviour of the nanofibrous mats were investigated in accordance with BS EN 13726-1:2002 section 3.7 dispersion and solubility of hydrogel dressings in water. The test was carried out in an environment similar to biological system – the temperature is controlled in 37°C and humidity is about 65%. The results obtained from this test provide an idea about the solubility of the fibres when these fibres install in a biological environment. The results show that the as-spun Na-alginate/PEO fibres dissolved and disappeared within 5 minutes whereas the chemically treated nanofibres remained intact even after 24 hours in water. This result confirms the realization of Ca-alginate fibres.

Conclusions

PEO-loaded smooth alginate nanofibres are produced using electrospinning technique. The as-spun sodium alginate/PEO nanofibres have been converted to silver loaded calcium alginate nanofibres by wet chemical treatment with calcium chloride and silver nitrate. Although the production rate and mass of the yielded fibres are limited the fibres can be incorporated with other construct to obtain advance functionalities. The chemical treatment and characterization process were done systematically. The results showed that the smooth and uniform nanofibres are produced from 4 wt. % of 70:30 Na-alginate/PEO polymer compositions. The insoluble alginate fibres were obtained by cross-linking the asspun Na-alginate/PEO nanofibres with calcium chloride, which is confirmed by solubility test. The results also revealed that the silver was successfully incorporated with post-treatment of Ca-alginate nanofibres, which was confirmed by EDX analysis. This tailor-made alginate nanofibrous mat can be used in developing tissue-engineered biomedical devices and in constructing interactive wound care materials. In wound dressing applications, this antibacterial alginate nanofibrous mat will be effective to mitigate bacterial infection as well as to promote the growth of epithelium cell in the wounds. The calcium ion in the chemically modified alginate nanofibres would interact with the sodium ion of wound fluids. As a result an ion exchange reaction can take place, which leads to the formation of sodium alginate gel that provides wound healing moisturous environment. Furthermore, it can be expected that the nanofibrous alginate dressing would be more effective and responsive in wound healing because of high conformability with sharp edges of wounds.

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