Macromolecular Research, Vol. 19, No. 4, pp 345-350 (2011) DOI 10.1007/s13233-011-0402-2

www.springer.com/13233

Preparation and Characterizations of Anisotropic Chitosan Nanofibers via Electrospinning R. Nirmala1, Baek Woo Il2, R. Navamathavan3, Mohamed H. El-Newehy4, and Hak Yong Kim*,2,4 1

Bio-nano System Engineering, College of Engineering, Chonbuk National University, Jeonju 561-756, Korea 2 Department of Textile Engineering, Chonbuk National University, Jeonju 561-756, Korea 3 School of Advanced Materials Engineering, Chonbuk National University, Jeonju 561-756, Korea 4 Petrochemical Research Chair, Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia Received July 13, 2010; Revised September 15, 2010; Accepted October 27, 2010

Abstract: We report the preparation of anisotropic chitosan nanofibers prepared using an electrospinning technique. The effect of electrospinning on the formation of nanofibers was examined from results of bulk chitosan. The morphological, structural characterizations and thermal properties of the chitosan bulk and electrospun nanofibers were analyzed by field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetry (TGA), Fourier transform infrared (FTIR), and Raman spectroscopy. Matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) was performed to accurately characterize the high aspect ratio nanofiber structure by the direct identification of mass resolved chains. FE-SEM showed that the electrospun chitosan nanofibers had diameters ranging from 10 to 1,200 nm with an anisotropic nature. MALDI-TOF revealed the presence of lower mass group protonated amino groups, which was the main constituent for the formation of the ultrafine nanofibers in chitosan. Keywords: electrospinning, chitosan, anisotropic, nanofibers, structure.

Introduction

nanofibers are influenced by the fabrication conditions such as molecular weight, and experimental parameters. Some studies determined the effect of the electrospinning process on chitosan nanofibers as well as blended with other polymers.10-18 Chitosan molecules are neutral and form intermolecular hydrogen bonds that make the dissolution of the polymer in water difficult. However, due to presence of amino groups in its chain, chitosan can be dissolved in an acidic aqueous solution and possesses properties of a cationic polyelectrolyte. The molecular weight of chitosan used in the electrospinning process needs to be very low since this polymer forms strong networks through the action of intermolecular and/or intramolecular hydrogen bonds. However, in terms of structural morphology, it is very important to make further improvements to electrospinning process, in order for them to be competitive to biomedical applications. Therefore, a deeper understanding of the physical processes taking place in electrospinning process of chitosan is of crucial importance. The anisotropic nanofibers can be utilized for controlled drug delivery and tissue culture scaffold in medical field and separation of metals owing to its high surface area-to-volume ratio. In this study, we focused on the formation of anisotropic high aspect ratio nanofibers via electrospinning and its ori-

Chitosan is a natural non-toxic biopolymer derived by the deacetylation of chitin, possessing unique polycationic, chelating, and film-forming properties due to the presence of active amino and hydroxyl functional groups. As a natural polymer, chitosan intrinsically exhibits enticing properties such as biocompatibility, biodegradability, antimicrobial activity, non-toxicity and its adequate absorption capabilities. Owing to these properties, chitosan is widely used in many different fields, including medicine, food and chemical engineering, pharmaceuticals, nutrition, and agriculture.1-6 Electrospinning of biologically significant polymers have increased since the electrospun membranes were identified as a candidate for tissue engineering constructs.7-9 The nanofibers produced by electrospinning process have showed excellent characteristics such as large surface area-to-volume ratio and high porosity with small pore size. Chitosan in the nanofiber mats format has a great potential to be widely used in various applications derived from its biocompatibility and biodegradability. The physical and chemical properties of these chitosan *Corresponding Author. E-mail: [email protected] The Polymer Society of Korea

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gin by performing the detailed structural characterizations. Though there are many reports available on electrospun chitosan nanofibers, however, there was no detailed study based on the formation of ultrafine nanofibers in chitosan. Therefore, we performed extensive analysis to elucidate the morphological, structural and chemical characterizations of the electrospun chitosan nanofibers. In order to better understand the reason for the formation of anisotropic nature of nanofibers in chitosan, we have carried out the comparative study with those of the chitosan bulk.

Experimental Chitosan powder (degree of deacetylation = 85%, low molecular weight, Wako Pure Chemical Industries, Japan) was used in making the solution. Chitosan nanofibers were electrospun from 7 wt% concentration of chitosan in trifluoroacetic acid/dichloromethane (MC) 80:20 v/v (Samchun Chemicals, Korea). Power supply of positive polarity was used to apply a voltage of 22 kV to the syringe micro-tip. The tip-to-collector distance was 15 cm. Polymer solution was fed to the 5 mL syringe with plastic micro-tip with a diameter of 0.3 mm. All experiments were performed at room temperature. The chitosan bulk and the electrospun mat were dried in vacuum and these samples were used for further characterizations. The morphology and diameter of the as-spun chitosan nanofibers were observed by using field-emission scanning electron microscopy (FE-SEM, Hitachi S-7400, Hitachi, Japan). Structural characterization was carried out by X-ray diffraction (XRD, Rigaku, Japan) operated with Cu-Kα radiation (λ = 1.540 Å). The bonding configurations of the samples were characterized with Fourier-transform infrared (FTIR) spectroscopy. Differential scanning calorimetry (DSC, Perkin-Elmer, USA) characterizations were performed for the chitosan bulk and the electrospun mats under nitrogen with a flow rate of 20 mL/min. The samples were heated from room temperature to 250 oC at a scanning rate of 10 oC/min. Thermogravimetric analysis (TGA, Perkin-Elmer, USA) were carried out for the samples under nitrogen with a flow rate of 20 mL/min. The samples were heated in a platinum pan from 30 to 800 oC at a rate of 10 oC/min and the differential thermogravimetry graph was recorded. The chitosan bulk and electrospun mat were analyzed by matrix-assisted laser desorption ionization time-offlight (MALDI-TOF, Voyager-DE STR Biospectrometry workstation, Applied Biosystems, USA) to determine the accurate mass values of chitosan.

Results and Discussion Figure 1 shows the SEM image of the chitosan bulk and the electrospun nanofibers. The raw chitosan powder displayed a dense surface (Figure 1(a)). The chitosan powder 346

Figure 1. SEM images of (a) chitosan bulk and (b) chitosan nanofibers.

was properly dissolved into the solvent to prepared nanofibers by electrospinning. These as-spun nanofibers exhibited smooth surface and bead-free uniform diameters along their lengths (Figure 1(b)). When a polymer solution is forced through a spinneret, the process aligns the chain in the direction of the extrusion with a similar alignment of the crystallites if the solid polymer is partially crystalline. During the electrospinning process, the chitosan nanofibers were collected on the rotating drum. The additional orientation that is typically imposed after the spinning causes additional alignment of the crystallites, and the stretching and alignment of the amorphous chains separating the crystallites.19 Figure 2(a) and 2(b) show the low and high magnification FE-SEM images of electrospun chitosan nanofibers, respectively. As shown in Figure 2, it was observed that the ultrafine nanofibers strongly bound within the main fibers. It was clearly visible that the formation of ultrafine nanofibers was strongly bound in between the main fibers. Our experiments revealed that the branching of ultrafine nanofibers from the main fibers, as shown in Figure 2. The split of ultrafine nanofiber that is apparent in the low mass group exist in the chitosan polymer, however, the original main fiber was continuous. In order for ideal non-branched fibers to be electrospun, an appropriate level of competition between electrical forces and surface tension must be maintained. Chitosan is ionic polyelectrolyte in which the reactive functional groups may yield reactions of chemical exchange when they are mixed with solvent. A higher charge density was exhibited on the surface of ejected jet form during elecMacromol. Res., Vol. 19, No. 4, 2011

Preparation and Characterizations of Anisotropic Chitosan Nanofibers via Electrospinning

Figure 3. Frequency distribution of anisotropic nature of electrospun chitosan nanofibers.

Figure 2. FE-SEM images of electrospun chitosan nanofibers (a) low and (b) high magnification.

trospinning. As the charges carried by the jet increase, higher elongation forces are imposed to the jet under the electrical field resulted in a branching, which is possibly due to the low molecular mass groups of the bulk chitosan. At this stage, the reactive ions in the solution drive further by the strong applied electric field. Then the solution become highly ionized state and forced to come out from the syringe can be branched as anisotropic structures in between the main fibers by relaxing the electrical stress. Further utilization of chitosan nanofibers that are electrospun from chitosan solutions in trifluoroacetic acid with dichloromethane as the modifying co-solvent is limited by the loss of the fibrous structure as soon as the nanofibers are in contact with neutral or weak basic aqueous solutions due to complete dissolution of the nanofibers. Dissolution occurs as a result of the high solubility in these aqueous media of -NH3+CF3COO- salt residues that are formed when chitosan is dissolved in solvent. Scheme I delineates the reaction that may occur during the neutralization of -NH3+CF3COO- salt residues along the chitosan chains.20,21 From the FE-SEM images, we calculated the frequency distribution of both main fibers and the high aspect ratio

nanofibers as shown in Figure 3. The diameter of the chitosan nanofibers were observed to be in the range of 150 to 1,200 nm, whereas the ultrafine structures consisted of regularly distributed very fine nanofibers with diameters of about 9 to 55 nm. The crystalline structures of as-electrospun chitosan nanofibers were characterized by XRD, and the result was compared with that acquired from the bulk which was used for electrospinning. The XRD pattern of the bulk and electro-

Figure 4. XRD patterns of chitosan bulk and electrospun nanofibers.

Scheme I. Possible chemical reaction that occur during neutralization of as electrospun chitosan nanofibers with trifluoroacetic acid/ dichloromethane. Macromol. Res., Vol. 19, No. 4, 2011

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Figure 6. Raman spectra of the chitosan bulk and electrospun nanofibers.

Figure 5. FTIR spectra of the chitosan bulk and and electrospun nanofibers.

spun mats of chitosan is shown in Figure 4. The diffraction pattern at 20o was very intense peak, indicating that the crystalline nature of bulk chitosan. A broad peak at about 19o was exhibited in the electrospun chitosan nanofibers mat which corresponds to the characteristic diffraction pattern for the chitosan polymer. This diffraction peak appears to be very broad due to the amorphous nature of electrospun chitosan nanofibers. The crystallinity of as electrospun chitosan nanofibers was less and the reflection peak was broader than for the bulk, suggesting that the sizes of chitosan crystallites were smaller in the electrospun nanofibers. FTIR spectroscopy was used to investigate the changes of the functional groups that occur during the chitosan nanofiber via electrospinning. Figure 5 shows the FTIR spectrum of the electrospun chitosan nanofibers and the bulk. The chitosan nanofibers exhibited a number of transmittance peaks corresponding to amide-I band at 1680 cm-1, amid-II band at 1535 cm-1, C-H stretching band at 2977 cm-1, the bridge oxygen stretching band at 1386 cm-1, and the C-O stretching bands at 1078 and 1206 cm-1.9,22 The broad peak between 3400 and 3700 cm-1 corresponds to a stretching of -OH and to physisorbed moisture on the surface in several modes. The intensity of amide-I is larger than that of the amide-II which is attributed to the resonance between the carbonyl stretching and the electric field of the incoming IR 348

beam. For comparison, we have measured FTIR spectrum of chitosan bulk as shown in Figure 5. Although the line shape of chitosan bulk follows the similar trend to that of the electrospun chitosan nanofibers, however, some of the chemical bonding configurations appeared to be mixed stretching groups. Because of chitosan has functional groups like hydroxyl, amines and amides, which can act as hydrogen bond acceptors or donors, it can be probably bonded or linked with hydrogen bond donors or acceptors compounds. To further understand the bonding structure of the electrospun chitosan nanofibers and bulk, we have performed Raman spectroscopy. Figure 6 shows the Raman spectra of the electrospun chitosan nanofibers and the bulk. The prominent Raman peaks can be assigned as the following; the CC stretching region (1050-1200 cm-1), the CNH bending region (1290-1350 cm-1), amide-III (1445 cm-1), amide-II (1550 cm-1) and amide-I (1635 cm-1). In comparing the Raman spectra of the chitosan nanofibers and the bulk, obviously, a slight difference was observed. The electrospun chitosan nanofibers exhibit weak trans-amide conformation (1310-1350 cm-1 and 1440-1490 cm-1), indicating that the amide region is in a gauche-conformation.23 The changes in crystallinity of both chitosan bulk and electrospun nanofibers upon thermal treatment were investigated by DSC at a heating rate of 10 oC/min, and the results are shown in Figure 7. For comparison, the chitosan bulk was also examined by DSC at the same heating rate. The DSC trace does not show any transition which could be attributed to the glass transition. The chitosan bulk and electrospun chitosan nanofibers showed an endothermic peak at 108 and 88 oC, respectively. The endothermic curve of chitosan nanofibers became broad and obtuse, and the peak shifted toward the lower temperature. This result indicated that the crystalline microstructure of electrospun fibers did not develop very well. This result is in good agreement with XRD data. However, the endothermic peak was disappeared Macromol. Res., Vol. 19, No. 4, 2011

Preparation and Characterizations of Anisotropic Chitosan Nanofibers via Electrospinning

Figure 7. DSC thermograms of chitosan bulk and electrospun nanofibers at a heating rate of 10 oC/min. Figure 9. MALDI-TOF mass spectrum of (a) chitosan bulk and (b) electrospun nanofibers, showing the resolved mass groups.

Figure 8. TGA graphs of the chitosan bulk and electrospun nanofibers.

during the second heating cycle as shown in Figure 7. This result indicated that the surface tension release was a significant contribution to the disappearance of the endothermic peak.24 The TGA results in Figure 8 provided quantitative information on the chitosan nanofibers functionalization. The TGA of chitosan revealed that the bulk and the electrospun nanofibers decomposed in a single step. However, the onset decomposition temperature for chitosan nanofibers was Macromol. Res., Vol. 19, No. 4, 2011

125 oC, while for chitosan bulk was 270 oC. The data obtained with this study demonstrated significant differences in the thermal stabilities between the starting physical form (chitosan bulk) and the electrospun chitosan nanofibers. Figure 9(a) and 9(b) show the MALDI-TOF mass spectrum of chitosan bulk and electrospun nanofibers, respectively. This data revealed that the hydrolyzed nature of the chitosan solution. The well-resolved peaks at lower molecular mass were attributed to the potential information on the structure of chitosan. Due to the presence of protonated amino groups chitosan exhibits polyelectrolyte character in trifluoroacetic acid/dichloromethane solution and its hydrodynamic behavior is intricate. As expected, the spectrum is dominated by a series of peaks corresponding to the lower mass groups which was the main constituent for the formation of the ultrafine nanofibers in chitosan. The MALDI spectrum is dominated by a series of intense peaks ranging from 20 to 450 with a small mass group. The structural identification of MALDI peaks was mainly assigned on the basis of empirical formula, therefore, it is suggested the possible mass groups. From this result it was clearly confirmed the presence of the lower molecular mass constituents of ultrafine nanofibers in chitosan were of hydroxyl, 349

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protonated amines and amide, hydrogen bonded compounds.

Conclusions Chitosan nanofibers with ultrafine structure were successfully produced by electrospinning technique. These as-spun nanofibers were observed to be smooth with uniform diameters along their lengths. Ultrafine chitosan nanofibers with diameters of about 9 to 55 nm were bound in between the main fibers. XRD pattern of the electrospun chitosan nanofibers exhibited a broad peak at 19o. The chitosan bulk and electrospun chitosan nanofibers showed a single melting peak almost at 125 and 270 oC, respectively. An endothermic peak was observed at 88 and 108 oC during the first heating cycle for electrospun chitosan nanofibers and the bulk, respectively. Characterization of the electrospun chitosan nanofibers via FTIR and Raman spectroscopy revealed that the amide groups terminated chitosan nanofibers with a stable structure. The MALDI-TOF spectrum was dominated by a series of peaks corresponding to the lower mass protonated amino groups which was the main constituent for the formation of the ultrafine nanofibers in chitosan. Acknowledgements. This work was supported by the grant of the Korean Ministry of Education, Science and Technology (The Regional Core Research Program/Center for Healthcare Technology & Development, Chonbuk National University, Jeonju 561-756, Republic of Korea).

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