Preparation and characterization of antibacterial alginate nanofibres

PREPARATION AND CHARACTERIZATION OF ANTIBACTERIALALGINATE NANOFIBRES

M. Forhad Hossain, R. Hugh Gong, Muriel Rigout

Abstract

Nanofibrous mats, produced from sodium alginate (Na-alginate), has

been successfully converted to insoluble antibacterial fibres.

Sodium alginate is a very popular thickening agent in textile

printing industry. In this study, however, this biopolymer has been

electrospun 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 anti-water and

antibacterial properties, these fibres are then chemically modified

by treating with CaCl2 and AgNO3 in ethanol absolute solution. During

chemical treatment, 1.0 wt.% of CaCl2 and 0.5 wt.% of AgNO3 were

used. The nanofibres structure and morphology were characterized by

field gun emission Scanning Electron Microscope (SEM), Energy

Dispersion X-ray (EDX), and Fourier Transform Infrared Spectroscopy

(FTIR).

Key words: electrospinning; sodium alginate; poly(ethylene oxide);anti-bacterial; nanofibres

1 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 (Qin 2008). It can be fabricated

as nanofibrous mats by electrospinning technique for biomedical or

healthcare applications. The as-spun Na-alginate/PEO nanofibres is

water soluble and is unable to sustain in aqueous environments which

might present in targeted biomedical applications. Thus, these

fibres need to be converted to insoluble to attain the structural

benefit of nanofibrous mats. Moreover, the most widely used

antibacterial agent, silver is incorporated with the alginate

nanofibres to gain antibacterial properties of nanofibres. This

silver-containing nanofibr can be used in precise biomedical

applications and wound care products. Some studies (Xu et al.

2006;Rujitanaroj et al. 2008;Son et al. 2006) revealed silver loaded

electrospun nanofibres from different biopolymers. They used silver

nanoparticles in the spinning solution. However, in this study, the

silver particles have been incorporated in the fibres by chemical

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 (Bhattarai et al.

2006;Kong et al. 2009;Lu et al. 2006) have been carried out to

convert Na-alginate nanofibres to insoluble Ca-alginate fibres.

These researchers made the Ca-alginate nanofibres by treating the

Na-alginate nanofibres with CaCl2 following a certain chemical

process. Le et al. (1997) demosntrated the chemical modification

process involved to produce Ag-, Na-, and Ca-alginates fibres in wet

spinning process. In this 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 (Le et al. 1997). It

is easy to incorporate silver ions onto the calcium alginate fibres

by treating with silver nitrate solution (Qin 2008). However, due to

pre-reaction of silver nitrate and calcium chloride in solution at

the mixing stage in ethanol absolute, in this project, the Na-

alginate/PEO nanofibres was treated with calcium chloride and silver

nitrate solution consecutively.

In this study, the chemical treatment comprises several subsequent

treatments to obtain insoluble alginate nanofibres. The first stage

of this process is to remove the PEO from the Na-alginate/PEO fibres

by soaking the fibres in ethanol absolute. The PEO component of the

fibres dissolves and is washed out, leaving Na-alginate nanofibres.

The Na-alginate is insoluble in ethanol but soluble in water. Thus,

the subsequent treatments are carried out in ethanol absolute

solution to obtain Ag-,Ca-alginate nanofibres. The following

reactions are taken place during this treatment process:

2Na-alginate + CaCl2 Ca(alginate)2 + 2NaCl

Na-alginate + AgNO3 Ag-alginate + NaNO3

2 Experimental procedures

2.1 Materials 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.

2.1.1 Preparation of treatment solution

To prepare 100gm 1.0 wt.% CaCl2 solution, 1.0 gm CaCl2 was added in

99.0gm ethanol absolute in a bottle. Then, the sealed bottle was

transferred on a shaker for gentle shacking at room temperature

(around 20°C) for overnight to obtain uniform solution. Similarly,

0.5 wt.% AgNO3 solution was prepared for carrying out the chemical

treatment process.

2.2 Treatment processA total 6 petri-dish were prepared in a fume cupboard to perform

this experiment. Na-alginate/PEO nanofibres were placed in a petri-

dish containing ethanol absolute solution (about 25 ml) to soak the

sample. The petri-dish was shacked gently for 10 minutes to dissolve

the PEO. The samples were then transferred in a petri-dish

containing 1.0 wt.% CaCl2 solution in ethanol absolute and stands for

10 minutes. At this stage, an ion exchange reaction would take place

between sodium ion (Na+) of Na-alginate nanofibres and calcium ion

(Ca2+) of CaCl2. As a result the Na-alginate nanofibres would convert

to Ca-alginate nanofibres. The samples were then transferred in

another petri-dish containing 0.5 wt.% AgNO3 solution, and stand for

10 minutes. At this stage, the Ca-alginate nanofibres would convert

to Ag-Ca-alginate nanofibres. Then, the samples were washed in

ethanol absolute solution to remove precipitated or unreacted salts.

Finally, the samples were dried at room temperature (around 20°C).

All of these experiments were carried out at room temperature.

2.3 Morphology and characterization of nanofibres The morphology and characterization of nanofibres mats was observed

by field gun emission 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 SEM-EDX observation,

the samples were 0.5cm×0.5cm in size and adhered on a specimen stub

by carbon tape specified for SEM purpose. Then the samples were

coated with carbon by using gatan Precision Etching Coating System

(model: 682). The SEM images were taken at 2000×, 10000× and 20000×

magnifications. The SEM operating parameters were set at 6kV

accelerating voltage and spot size 3. The fibres diameters were

manually measured by using the line-drawing feature in ImageJ

(ImageJ 2004) 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 SEM image scale bar. For EDX analysis the

scanning was taken at 2000× magnification and the parameters were

set at 10kV accelerating voltage and spot size 3.

The chemical structure of Na-alginate/PEO, chemically modified

alginate fibres, were analysed by FTIR. The infrared spectral of

absorption mode of the nanofibres surface is obtained from FTIR

spectrometer which is connected with a PC. The spectra were scanned

with a spectral range of 4000-400cm-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 (2cm*2cm) 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.

3 Results and discussion

3.1 Morphology and characterization of nanofibres The chemical treatments of the Na-alginate/PEO nanofibres were

carried out with AgNO3 and CaCl2 in ethanol solution. The effect of

chemical treatment on morphology was seen by SEM images and EDX

analysis was carried out to examine the presence of the expected

elements. Due to chemical treatment the morphology of the

nanofibrous mesh is changed significantly – a lot of spherical

particles are created in the fibres. These particles clearly

represent the presence of silver (Ag).

The figures 1[A1][A2] show the smooth electrospun nanofibres with

126nm average diameter yielded from Na-alginate/PEO. The figures

1[B1][B2] show that the silver particles have adhered to the

alginate fibres and fibre morphology is changed considerably. The

average diameter of silver particles was 210 nm with good size

distribution (Figure 1[B2]). However, the uniformity of the fibres

deteriorated due to chemical treatment, some thick and thin places

have appeared in the fibres.

A1 A2

B1 B2

Mean=210;SD=32;Min=146;Max=279Min=146;Max=279

Figure 1: SEM images ×10K of 70:30 Na-alginate/PEO nanofibres and fibres size distribution before chemical treatment [A1][A2]; and silver particles size distribution of in the chemically treated alginate nanofibres [B1][B2].

3.2 EDX analysisThe EDX assessment was done to examine the presence of prime

elements such as silver (Ag), sodium (Na), calcium (Ca), oxygen (O)

and chlorine (Cl) in the fibres. In the sample treated with 1.0 wt.%

CaCl2 and 0.5 wt.% AgNO3, the %wt. of silver (Ag), calcium (Ca),

sodium (Na), chlorine (Cl), and oxygen (O) was found to be 28.07,

15.36, 11.83, 27.41 and 17.3, respectively. Figure 2 shows the

relative amount of Ag, Ca, Na, O, and Cl available in the fibres.

Thus, the antibacterial agent (silver) is successfully incorporated

in the alginate nanofibres.

silver calcium sodiumchlorineoxygen051015202530 28.07

15.3611.83

27.41

17.3

% wt.

Figure 2 Relative amount of silver present in the nanofibres

3.3 FTIR analysisFourier Transform Infrared Spectroscopy (FTIR) was used to observe

the spectral peak variation between the Na-alginate/PEO and post-

treatment alginate nanofibres. Due to the chemical treatment, the

peaks between the Na-alginate/PEO fibres and treated fibre are

changed. The peak at 2883 cm-1 in 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 Figure 3

shows the peaks for –OH group occurred at 3346cm-1 for

Na-alginate/PEO, is moved to 3336 cm-1 for chemically modified

alginate fibres. Due to the chemical treatment, Na-alginate

nanofibres is turned into calcium alginate nanofibres and this is

confirmed as the frequencies shifted to 3336 cm-1 from 3346 cm-1 of

Na-alginate/PEO nanofibres (Figure 3).

4000 3500 3000 2500 2000 1500 1000 500

Absorbence

W avenumber (cm -1)

B A

3400 3200 3000 2800 2600 2400 2200 2000

Absorbence

W avenumber (cm -1))

B A

Figure 3 The FTIR spectra of Na-alginate/PEO nanofibres before chemical treatment (A), and after chemical treatment (B).

3.4 Solubility of nanofibres

Insolubility of the nanofibres is important to exploit the

functional benefits from nanofibrous structure fully. When the

nanofibres is used in an aqueous environment it should retain the

structure to attain these structural benefits. According to BS EN

13726-1:2002 method, the nanofibres in the petri-dish were assessed

visually. The results show that the fibres remains intact even after

24 hours in water. Thus, the Na-alginate nanofibres have been

converted to insoluble and non-dispersible alginate nanofibres.

4 Conclusions

By using electrospinning technique, sodium alginate is transformed

to nanofibres followed by chemical treatment with calcium chloride

and silver nitrate to obtain calcium alginate nanofibres with

antibacterial properties. The results reveal that the silver is

successfully incorporated with post-treatment of Ca-alginate

nanofibres, and this is confirmed by EDX analysis. Silver loaded

biocompatible and biodegradable, and non-toxic alginate nanofibres

is produced with low mean fibre diameter and narrow fibre size

distribution. The tailor-made alginate nanofibre mats can be used in

novel applications such as drug delivery, interactive wound

dressing, biofilm and filtration.

References

Bhattarai, N., Li, Z. S., Edmondson, D. & Zhang, M. Q. (2006). Alginate-based nanofibrous scaffolds: Structural, mechanical, and biological properties. Advanced Materials, 18(11), 1463-+.

ImageJ. (2004). ImageJ, National Institutes of Health, United States [Online]. Available: http://rsb.info.nih.gov/ij/index.html [Accessed 08/01/14].

Kong, Q. S., Yu, Z. S., Ji, Q. & Xia, Y. Z. (2009). Electrospinning of Sodium Alginate with Poly(ethylene oxide), Gelatin and Nanometer Silver Colloid. Materials Research, Pts 1 and 2, 610-613, 1188-1191.

Le, Y., Anand, S. C. & Horrocks, A. R. (1997). Using alginate fibresas drug carrier for wound dressing. Medical Textiles '96. Cambridge: Woodhead publishing.

Lu, J.-W., Zhu, Y.-L., Guo, Z.-X., Hu, P. & Yu, J. (2006). Electrospinning of sodium alginate with poly(ethylene oxide). Polymer, 47(23), 8026-8031.

Qin, Y. (2008). Alginate fibres: an overview of the production processes and applications in wound management. Polymer International, 57(2), 171-180.

Rujitanaroj, P.-o., Pimpha, N. & Supaphol, P. (2008). Wound-dressingmaterials with antibacterial activity from electrospun gelatinfiber mats containing silver nanoparticles. Polymer, 49(21), 4723-4732.

Son, W. K., Youk, J. H. & Park, W. H. (2006). Antimicrobial cellulose acetate nanofibers containing silver nanoparticles. Carbohydrate Polymers, 65(4), 430-434.

Xu, X., Yang, Q., Wang, Y., Yu, H., Chen, X. & Jing, X. (2006). Biodegradable electrospun poly(l-lactide) fibers containing antibacterial silver nanoparticles. European Polymer Journal, 42(9),2081-2087.