Biotechnology Advances

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Research review paper

Novel chitin and chitosan nano ﬁbers in biomedical applications

R. Jayakumar

^a,

⁎ , M. Prabaharan

^b

, S.V. Nair

^a

, H. Tamura

^c

aAmrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidhyapeetham University, Cochin-682 041, India

bDepartment of Chemistry, Faculty of Engineering and Technology, SRM University, Kattankulathur-603 203, India

cFaculty of Chemistry, Materials and Bioengineering and High Technology Research Centre, Kansai University, Osaka-564-8680, Japan

a b s t r a c t a r t i c l e i n f o

Article history:

Received 2 August 2009

Received in revised form 27 October 2009 Accepted 4 November 2009

Available online 11 November 2009

Keywords:

Chitin Chitosan Nanoﬁbers Tissue engineering Drug delivery Biosensors Wound healing Filtration

Chitin and its deacetylated derivative, chitosan, are non-toxic, antibacterial, biodegradable and biocompatible biopolymers. Due to these properties, they are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressings, separation membranes and antibacterial coatings, stent coatings, and sensors. In the recent years, electrospinning has been found to be a novel technique to produce chitin and chitosan nanofibers. These nanofibers find novel applications in biomedical fields due to their high surface area and porosity. This article reviews the recent reports on the preparation, properties and biomedical applications of chitin and chitosan based nanofibers in detail.

Contents

1. Introduction . . . 142

2. Electrospinning of chitin and chitosan . . . 143

2.1. Electrospinning of chitin . . . 143

2.2. Electrospinning of chitosan . . . 143

3. Applications of chitin and chitosan nanoﬁbers . . . 146

3.1. Filtration . . . 146

3.2. Biosensors . . . 146

3.3. Wound dressing . . . 146

3.4. Tissue engineering . . . 147

3.5. Drug delivery . . . 148

4. Conclusions . . . 148

Acknowledgments . . . 149

References . . . 149

1. Introduction

Chitin, the second most abundant natural polysaccharide, is synthesized by a number of living organisms. Chitin occurs in nature

as ordered microﬁbrils, and is the major structural component in the exoskeleton of arthropods and cell walls of fungi and yeast. The main commercial sources of chitin are crab and shrimp shells, which are abundantly supplied as waste products of the seafood industry.

Because chitin is not readily dissolved in common solvents, it is often converted to its more deacetylated derivative, chitosan (Kurita, 2001;

Rinaudo, 2006; Pillai et al., 2009). Chitosan is often identiﬁed by its degree of deacetylation (DD), a percentage measurement of free amine groups along the chitosan backbone (Roberts, 1992). Because

⁎ Corresponding author. Tel.: +91 484 2801234; fax: +91 484 2802020.

E-mail addresses:rjayakumar@aims.amrita.edu,jayakumar77@yahoo.com (R. Jayakumar).

doi:10.1016/j.biotechadv.2009.11.001

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of its solubility in acidic, neutral and alkaline solutions, chitosan is preferred over chitin for a wide range of applications.

Chitin and chitosan are biocompatible, biodegradable, and non- toxic, and are anti-microbial and hydrating agents. Chitin and chitosan are easily processed into gels (Nagahama et al., 2008a,b), membranes (Jayakumar et al., 2005, 2007, 2009; Madhumathi et al., 2009a,b), nanoﬁbers (Schiffman and Schauer, 2007a; Shalumon et al., 2009), beads (Jayakumar et al., 2006), microparticles (Prabaharan and Mano, 2005), nanoparticles (Anitha, 2009), scaffolds (Madhumathi et al., 2009c; Maeda et al., 2008) and sponges (Muramatsu et al., 2003;

Portero et al., 2007) forms. There are a number of promising applications of nanoscale thinﬁlms and ﬁbers based on chitin/chitosan (Wang and Hon, 2003; Rinaudo, 2006; Pillai et al., 2009).

Recently, much attention has been paid to electrospinning process as a unique technique because it can produce polymer nanofibers with diameter in the range from several micrometers down to tens of nanometers, depending on the polymer and processing conditions. In electrospinning, a high voltage is applied to create electrically charged jets of a polymer solution. These jets dry to form nanofibers, which are collected on a target as a non-woven fabric. These nanofibers are of considerable interest for various kinds of applications, because they have several useful properties such as high specific surface area and high porosity. Nanofibers containing chitin or chitosan yield potential applications in areas such asfiltrations, recovery of metal ions, drug release, dental, tissue engineering, catalyst and enzyme carriers, wound healing, protective clothing, cosmetics, biosensors, medical implants and energy storage (Zhang et al., 2005a,b; Fang et al., 2008).

In this review, we are reporting the different methods of preparation of electrospun chitin and chitosan nanoﬁbers and their application in biomedicalﬁelds in detail.

2. Electrospinning of chitin and chitosan 2.1. Electrospinning of chitin

Chitin is insoluble in most of the organic solvents. Due to its insolubility, its applications are limited in many applications. Chitin dissolves only in specific solvents such as N, N-dimethylacetamide (DMAC)-LiCl (Cho et al., 2000), hexafluoroacetone, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Kurita, 2001) and saturated calcium solvent (Jayakumar and Tamura, 2008; Nagahama et al., 2008a,b).Min et al.

(2004)performed electrospinning of chitin using HFIP solvent. Before electrospinning the chitin was depolymerized by gamma radiation to improve the solubility. The nanoﬁbers prepared by this method had the diameter less than 100 nm. Nanocompositeﬁber mats composed of electrospun poly(vinyl alcohol) (PVA) containingα-chitin whiskers prepared from shells of Penaeus merguiensis shrimps (Junkasem et al., 2006). A maximum tensile strength i.e., 5.7 ± 0.6 MPa was obtained when the chitin whisker to PVA ratio was approximately 5.1%. After this point, increasing the chitin content decreased the strength of the mats.

Electrospun chitin/poly(glycolic acid) (PGA) blend nanoﬁbers in HFIP was investigated to fabricate biodegradable and biomimetic nanostructured scaffolds for tissue engineering (Park et al., 2006a).

PGA was chosen because it was a biocompatible and biodegradable polymer. The PGA/chitin blend fibers have average diameters of around 140 nm, and their diameters have a distribution in the range 50–350 nm. In vitro degradation studies demonstrated that the blend fibers degraded faster than pure PGA fibers in phosphate buffer solution at pH 7.2. Electrospun chitin/silkfibroin (SF) nanofibers in HFIP solvent were also reported (Park et al., 2006b). The average diameters of chitin/SF blendfibers decreased from 920 to 340 nm with the increase of chitin content in blend compositions.Shalumon et al. (2009)developed a novel electrospun water-soluble carboxymethyl chitin (CMC)/PVA blend by electrospinning technique. In this study, the concentration of CMC and PVA in the blend was optimized.

Fibers prepared in this study were made water insoluble by cross- linking with glutaraldehyde vapors followed by thermal treatment.

Table 1 summarizes the experimental conditions adapted for the fabrication and obtained average size of electrospun chitin nanoﬁbers.

2.2. Electrospinning of chitosan

Chitosan is soluble in most of the acids. The protonation of chitosan changes it into a polyelectrolyte in acidic solutions. There are a few reports on polyelectrolytes that have successfully been electrospun (Duan et al., 2004). It has been theorized byMin et al.

(2004)that the repulsive forces between ionic groups within polymer backbone that arise due to the application of a high electricfield during electrospinning restrict the formation of continuousfibers and often produce particles. There were some works reported (McKee and Elkins, 2004; McKee et al., 2006) on developing an empirical equation forfiber diameter and the effects that the inter-molecular associations state of a polyelectrolyte have on electrospinning.

An electrospun non-woven fabric of chitosan was successfully prepared byOhkawa et al. (2004). This study focuses on the effect of the electrospinning solvent and the chitosan concentration on the morphology of the resulting non-woven fabrics. The solvents tested were diluted hydrochloric acid, acetic acid, formic acid and trifluoroacetic acid (TFA). As the chitosan concentration was increased, the morphology of the deposition on the collector changed from spherical beads to interconnectedfibrous networks. The addition of dichloromethane to the chitosan–TFA solution has improved the homoge- neity of the electrospun chitosanfiber. Under optimized conditions, homogenous chitosanfibers with a mean diameter of 330 nm were obtained. Ohkawa et al. (2004) proposed that TFA is the main constituent in the successful solvent system for chitosan because the amino groups of the chitosan can form salts (Hasegawa et al., 1992) with the TFA, which can effectively destroy the rigid interactions between the chitosan molecules thus facilitating electrospinning.

Similarly, Haider and Park (2009) developed electrospun chitosan nanofibers for absorbing metal ions. A follow-up study was conducted byOhkawa et al. (2006), which focused on idealizing the viscosity of chitosan solutions (Baumgarten, 1971; Fridrikh et al., 2003) in order to decrease the averagefiber diameter. It was determined that fiber diameter and polymer concentration have an inverse relationship. In another study,Homoyoni et al. (2009)developed electrospinning of chitosan. The problem of chitosan high viscosity, which limits its spinability, is resolved through the application of an alkali treatment which hydrolyzes chitosan chains and so decreases their molecular weight. The alkali treated chitosan in aqueous 70–90% acetic acid produces nanofibers with appropriate quality and processing stability.

Decreasing the acetic acid concentration in the solvent increases the mean diameter of the nanoﬁbers. Optimum sizes of nanoﬁbers were achieved with chitosan, which was hydrolyzed with alkali for 48 h.

The nanoﬁbers prepared from hydrolyzed chitosan resulted 74%

greater moisture regain than that prepared from untreated chitosan.

The diameter of these nanoﬁbers was strongly affected by the electrospinning conditions as well as the concentration of the solvent.

Chemical, structural, and mechanical analyses of chitosan nanofibers cross-linked with glutaraldehyde were conducted bySchiffman and Schauer (2007a). The solubility of chitosan nanofibers was greatly improved after cross-linking. The medium molecular weight chitosan nanofibers had a Young's modulus of 154.9±40.0 MPa and displayed a pseudo-yield point that arose due to the transition from the pulling of a fibrous mat with high cohesive strength to the sliding and elongation offibers. As-spun mats were found to be highly soluble in acidic and aqueous solutions. After cross-linking, the medium molecular weight fibers increased in diameter by an average of 161 nm and decreased Young's modulus of 150.8 ±43.6 MPa. Schiff base cross-linked chitosanfibrous mats were produced by a one-step electrospinning process that is 25 times faster and, therefore, more

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economical than a previously reported two-step vapor-cross-linking method (Schiffman and Schauer, 2007b). Theseﬁbrous mats were insoluble in acidic, basic, and aqueous solutions for 72 h. This improved production method results in a decreased averageﬁber diameter, which measures 128 ± 40 nm.

Further utilization of chitosan nanofibrous membranes that were electrospun from chitosan solutions in trifluoroacetic acid (TFA) with or without dichloromethane (DCM) as the modifying cosolvent was found to be limited by the loss of thefibrous structure when the membranes were in contact with neutral or weak basic aqueous solutions due to complete dissolution of the membranes (Sangsanoh and Supaphol, 2006). Much improvement in the neutralization method was achieved with the saturated Na2CO3 aqueous solution with an excess amount of Na2CO3in the solution. They reported that electrospun chitosan nanofibrous membranes, after neutralization in the Na₂CO₃aqueous solution, could maintain itsfibrous structure even after continuous submersion in phosphate buffer saline (pH = 7.4) or distilled water for 12 weeks.

Torres-Giner et al. (2008a,b) developed electrospun chitosan nanofibers using TFA and dicholomethane solvent. In addition,Torres- Giner et al. (2008a,b) also developed porous electrospun chitosan nanofibers using pure trichloromethane solvent. Besides electrospinning chitosan in TFA and trichloromethane, the second solvent system that has been demonstrated to effectively produce nanofibers is concentrated acetic acid.Geng et al. (2005)attempted to electrospin three kinds of demineralized and deproteinized chitosan powders.

However, uniformﬁbers were only fabricated from 7% chitosan in 90%

aqueous acetic acid solutions. It was noted that the surface tension and charge density were the key factors in determining the spinnability of the system. In the preliminary attempts to produce chitosan nanoﬁbers for wound or alternate medical applications, or the removal of metals from solutions for environmental applications, Vrieze et al. (2007) conducted a feasibility study concerning the electrospinning of chitosan in formic, acetic, lactic and hydrochloric acids. It was determined that chitosan can produce theﬁbers only with the aqueous acetic acid (Li and Hsieh, 2006; Sangsanoh and Supaphol, 2006).

Recently, electrospun chitosan composite nanofibrous mats have been fabricated using chitosan and synthetic biodegradable polymers such as poly (vinyl alcohol) (Miya et al., 1984; Zheng et al., 2001), poly (ethylene oxide) (PEO) (Yilmaz et al., 2003), poly(vinyl pyrrolidine) (PVP) (Ignatova et al., 2007), poly(lactic acid) (PLA) (Peesan et al., 2006; Torres-Giner et al., 2008a,b) and poly(ethylene terpthalate (PET) (Jung et al., 2007). These composite fiber mats are more advantageous over the electrospinning of pure chitosan because, the mechanical, biocompatible, antibacterial and other properties of the chitosan nanofibers were drastically enhanced by the addition of PVA, PEO, PLA, PVP and PET.

PVA is used for a variety of biomedical applications such as bone implants (Allen et al., 2004; Zhang et al., 2005a,b), and artificial organs (Chen et al., 1994). Several researchers have developed nanofibers of PVA with chitosan by electrospinning because PVA has good fiber- forming characteristics (Li and Hsieh, 2006; Zhou et al., 2006; Huang et al., 2007; Jia et al., 2007; Zhou et al., 2007). The electrospun composites of chitosan/PVA nanofibers in different ratios were reported for Table 1

Electrospun chitin and chitosan nanoﬁbers.

Polymer Solvent Degree of deacetylation (%) Averageﬁber diameter (nm) Reference

Chitin HFIP 8 110 Min et al. (2004)

Chitin HFIP 8 163 Noh et al. (2006)

Chitin/PGA HFIP 8 140 Park et al. (2006a)

Chitin/SF HFIP 8 340 Park et al. (2006b)

CMC/PVA Water 28.7 300 Shalumon et al. (2009)

Chitosan10 TFA/MC 78 330 Ohkawa et al. (2004)

Low MW chitosan TFA 74 74 ± 28 Schiffman and Schauer (2007a)

Medium MW chitosan TFA 83 77 ± 29

High MW chitosan TFA 72 108 ± 42

Practical grade chitosan TFA 75 58 ± 20

Medium MW chitosan TFA/GA 83 128 ± 40 Schiffman and Schauer (2007b)

Chitosan TFA/MC 95 130 ± 10 Sangsanoh and Supaphol (2006)

Chitosan aq AA 54 130 Geng et al. (2005)

Chitosan aq AA 75–85 70 ± 45 Vrieze et al. (2007)

Hexanoyl chitosan Chloroform 88 640–3930 Neamnark et al. (2006)

PEGylated chitosan DMF/THF 84.7 40–360 Du and Hsieh (2007)

Chitosan/PVA aq AA 90 106 ± 27 Duan et al. (2006)

Chitosan/PVA aq AA 82.5 100 ± 20 Li and Hsieh (2006)

Chitosan/PVA aq AA 90 80–150 Huang et al. (2007)

Chitosan/PEO aq AA 82 80 ± 35 Ojha et al., 2008

Chitosan/PEO aq AA 85 20–120 Bhattarai et al. (2005)

Chitosan/PEO AA 80 10–240 Kriegel et al. (2009a,b)

Chitosan/UHMWPEO (20%) aq AA/DMSO N85 102 ± 14 Zhang et al. (2008a,b)

Chitosan/UHMWPEO (10%) 138 ± 15

Chitosan/UHMWPEO (5%) 114 ± 19

Chitosan/PET TFA 85 500–800 Jung et al. (2007)

Chitosan/SF FA 86 180–790 Park et al. (2004)

Chitosan/HAp AA/DMSO 88 100–700 Yang et al. (2008)

Chitosan/P(LLA-CL) aq AA 80 400–2000 Chen et al. (2008a,b,c)

O-CMCS/PEO Water – 118 ± 41 Vondran (2007)

O-CMCS/PVA Water 84.7 130 Du and Hsieh (2008)

N-CECS/PVA aq AcrA – 100–420 Mincheva et al. (2007)

N-CECS/PVA Water 82.5 131–456 Zhou et al. (2008)

QCS/PVP Water 80 2440 ± 640 Ignatova et al. (2007)

QCS/PVA aq AA 80 60–200 Ignatova et al. (2006)

Chitosan-g-PEG/PLGA DMF/THF 85 Jiang et al. (2004)

PLGA-Chitosan/PVA THF-DMF-2% AA 90 275 ± 175 Duan et al. (2006)

Abbreviations: CMC, carboxymethyl chitin; PGA, poly(glycolic acid) (PGA), UHMWPEO, ultrahigh-molecular-weight poly(ethylene oxide); PET, poly(ethylene terephthalate); HAp, hydroxyapatite; P(LLA-CL), poly(L-lactic acid-co-ε-caprolactone); CMCS, carboxymethyl chitosan; CECS, carboxyethyl chitosan; PVA, poly(vinyl alcohol); QCS, quaternized chitosan;

PVP, poly(vinyl pyrrolidone); SF, silkfibroin; DMF, dimethylformamide; THF, tetrahydrofuran; GA, glutaraldehyde; TFA, trifluoroacetic acid; aq AA, aqueous acetic acid solution; FA, formic acid; MC, methylene chloride; aq AcrA, aqueous acrylic acid solution; TCM, trichloromethane; DMSO, dimethyl sulfoxide; HFIP, 1,1,1,3,3,3,-hexafluoro-2h-propanol.

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biomedical applications (Ding et al., 2002; Koski et al., 2004; Ohkawa et al., 2004; Zhang et al., 2005a,b; Lin et al., 2006; Zhang et al., 2007;

Zhou et al., 2008). Chitosan can be used as a thickener to improve the rheological properties of an aqueous solution to be electrospun because chitosan is compatible with other biocompatible polymers such as PVA (Miya et al., 1984; Zheng et al., 2001), PEO (Yilmaz et al., 2003) etc. A common defect observed when electrospinning is the formation of beads. To overcome this, additives such as salts (Fong et al., 1999) or surfactants (Lin et al., 2004) are added. Alternatively, the addition of cationic and anionic polyelectrolytes (Son et al., 2004) would also increase the conductivity of a solution and thus decreaseﬁber diameter.

Since chitosan is a linear cationic polymer, it was determined that chitosan can act like other ionic additives and reduceﬁber diameter and thus producing thinner, uniform, bead freeﬁbers (Lin et al., 2006; Jia et al., 2007).

Thermally cross-linked electrospun ﬁbers were prepared from chitosan/PVA in aqueous acrylic acid solutions (Zhou et al., 2006;

2007). To allow for utilization of the chitosan/PVA mats in a variety of applications, TEGDMA was added with chitosan/PVA solution to improve the mechanical properties of the resultingﬁbers. It was found that by adding TEGDMA prior to spinning followed by heat-treating the as-spun mats for 2 h at 80 °C cross-linkedﬁbrous mats could be fabricated.

The development of bioinspired or biomimetic materials is essential and has formed one of the most important paradigms in today's tissue engineering research.Zhang et al. (2008a) reported novel biomimetic nanocomposite nanoﬁbers of hydroxyapatite (HAp)/chitosan by combining an in situ co-precipitation synthesis approach with an electrospinning process. A model HAp/chitosan with the HAp mass ratio of 30 wt.% was also synthesized through the co-precipitation method so as to attain homogenous dispersion of the spindle-shaped HAp nanoparticles within the chitosan matrix.

Similarly, biocomposite nanoﬁbers were also prepared using of HAp with chitosan/PVA for biomedical applications (Yang et al., 2008).

PEO is also a biocompatible polymer (Grifﬁth, 2000) that has been used as a wound dressing (Yoshii et al., 1999) and cartilage tissue repair (Sims et al., 1996; Subramanian et al., 2005). The PEO had previously been electrospun (Doshi and Reneker, 1995; Fong et al., 1999; Deitzel et al., 2001a,b) with chitosan.Duan et al. (2004)electrospun chitosan/PEO and the results showed that PEO aided in the electrospinning of silk and collagen (Huang et al., 2001; Jin et al., 2002). When there was a mass ratio of PEO/chitosan of 2/1 or 1/1, the improved conductivity, surface tension, and solution viscosity of PEO/

chitosan greatly improved the electrospinning.Spasova et al. (2004);

Duan et al. (2004) and Desai et al. (2009)also reported the electrospinning of chitosan/PEO system. It was observed that as the proportion of chitosan increased, theﬁber diameter was decreased.

Bhattarai et al. (2005)developed electrospun chitosan with PEO. It was found that a chitosan/PEO ratio of 9/1 was an appropriate condition for the preparation of nanofibers for bone tissue engineering because thesefibers retained good structural integrity in water and promoted good adhesion of chondrocyte and osteoblast cells. In addition,Vondran (2007)has also studied the mechanical properties of chitosan/PEO nanofibrous mat as well as mats that were cross- linked by glutaraldehyde vapor. Chitosan/PEO nanofibers with surfactants were also reported for thefiltration applications (Kriegel et al., 2009a,b).

Zhang et al. (2008b)demonstrated the preparation of nanofibers by introducing an ultra high-molecular-weight PEO (UHMWPEO) into aqueous chitosan solution. This system produced the chitosan nanofibers with good structural stability and handling properties. Because of the excellent electrospinnability of this system, it was able to electrospin both the extremely thin nanofibers (b100 nm in diameters) and large microfibers (few tens of micrometers in diameters), which have significant implications in developing biomimetic and bioactive 3-D cell-scaffold complex for engineering tissues. The results suggest that

current eco-friendly and easily electrospinnable chitosan formulation could provide great potential for robust and scale-up production of the chitosan nanoﬁbers for efﬁcient practical applications in wound dressings, tissue engineering, drug delivery, and other industrial uses.

PET is used in the textile and plastic industry, its antibacterial properties have been widely studied (Huh et al., 2001; Yang et al., 2002).

Jung et al. (2007)reported that the chitosan/PET nanofibers were useful for medical applications. Chitosan/PET and chitin/PET were electrospun in a TFA/HFIP solution. Antibacterial activity studies indicated that chitosan/PET nanofibers inhibited the growth of bacteria much more effectively than both the pure PET and the chitin/PETfibrous mats.

Torres-Giner et al. (2008a,b) also developed chitosan/PLA blend nanoﬁbers by electrospinning in TFA and tricholoromethane mixture.

Collagen has been used previously for electrospinning using HFIP solvent (Matthews et al., 2002, 2003; Rho et al., 2006). To develop a better biomimetic extracellular matrix for the tissue engineering of functional biomaterials, a matrixfibrous mat of chitosan/collagen was developed (Chen et al., 2007, 2008a,b,c).Mo et al. (2007)reported the electrospun chitosan/collagen in HFIP/TFA. Composite nanofibrous membranes of type I collagen, chitosan, and polyethylene oxide were fabricated by electrospinning, which could be further cross-linked by glutaraldehyde vapor (Chen et al., 2008a,b,c). In this study, nanofiber diameter was found to be 134 ± 42 nm, which increased to 398 ± 76 nm after cross-linking. It was found that the Young's modulus of nanofiber was increased after cross-linking. However, the ultimate tensile strength, tensile strain, and water sorption capability were found to be decrease after cross-linking.

Zein is a relatively straightforward biopolymer to electrospin (Miyoshi et al., 2005; Torres-Giner et al., 2008a,b). It has low toxicity and therefore widely used in a broad range of areas, such as the food, pharmaceutical, and biodegradable plastics industry (Corradini et al., 2006). Torres-Giner et al. (2009) developed novel anti-microbial ultrathin structures of zein/chitosan blend by electrospinning for biomedical applications.

Silk fibroin is the fibrous protein that forms the filaments of silkworm silk. Due to its biocompatibility, biodegradability, low in- flammatory responses, and good oxygen and water vapor permeabil- ity, it has been used for several biomedical applications (Santin et al., 1999; Park et al., 2001). The novel biomimetic nanofibrous scaffolds were prepared using chitosan/SF in formic acid (Park et al., 2004).

While a pure chitosan system could not be electrospun, up to a 30/70 chitosan/SF in formic acid could be spun. The inﬂuence of a methanol treatment on the secondary structure of as-spun SF versus chitosan/SF was also investigated.

Recently, nanofibers based on chitosan derivatives have also been prepared for biomedical applications. Carboxymethyl, carboxyethyl and hexanoyl chitosan (Vondran, 2007; Mincheva et al., 2007; Peesan et al., 2006; Neamnark et al., 2006) derivatives were used for the preparation of nanofibers. Hexanoyl chitosan was used for medical applications since it has been proven to be resistant to hydrolysis by lysosome (Lee et al., 1995) and is anti-thrombogenic (Hirano and Noishiki, 1985).Neamnark et al. (2006)prepared hexanoyl chitosan nanofibers in chloroform solvent. The prepared nanofibers displayed ribbon-like morphology with diameters ranging from 0.64 to 3.93 µm.

O-Carboxymethyl chitosan (O-CMCS) is a water-soluble derivative of chitosan (Muzzarelli et al., 1994; Chen and Park, 2003). It has good moisture retention, gel-forming capability and antibacterial property, thus making it a good biomaterial (Chen et al., 2002, 2006).Vondran (2007)has prepared electrospun compositeﬁbrous mats using CMCS/

PEO using water. Thefibers appear to be continuous and cylindrical, while some beading was observed. The averagefiber diameter was found to be 118.19 ± 40.48 nm. Similarly, Du and Hsieh (2008) prepared O-CMCS blended PVA, PEO and poly(acrylic acid) (PAA) nanofibers by electrospinning. The optimal fiber formation was observed at equal mass composition of O-CMCS (89 kDa at 0.36 DS) and PVA. Thesefibers had an average diameter of 130 nm. Heat-

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induced esterification (at 140 °C for 30 min) produced inter-molecular covalent cross-links within and among fibers, rendering the fibrous membrane water-insoluble. Membranes containing higher O- CMCS carboxyl to PVA hydroxyl ratio retained betterfiber morphology upon extended water exposure, indicating more favorable intermolecular cross-links. Thefibrous membranes generated with less substituted O-CMCS were more hydrophilic and retained a greater extent of the desirable amine functionality.

Bicomponent nanofibers of N-carboxyethyl chitosan (N-CECS) and PVA were obtained by electrospinning (Mincheva et al., 2007). The electrospinning of N-CECS containing nanofibers was enabled by the ability of PVA to form an elastically deformable entanglement network based on hydrogen bonds. The average diameters of the bicomponentfibers were in the range of 100–420 nm. The average fiber diameter was tuned by varying the applied field strength and the N-CECS content in the spinning solution. The N-CECS/PVA nanofibrous mats were easily cross-linked by thermal treatment resulting in water-resistant materials, which retain theirfibrous structure upon one-week contact with water. In addition, biocomposite nanofibers were also prepared using of HAp with N-CECS/PVA for biomedical applications (Yang et al., 2008).

Novel quaternized chitosan (QCS)/PVA blend nanofibers were developed for wound-healing applications (Ignatova et al., 2006). The averagefiber diameter was found to be in the range of 60–200 nm. UV irradiation of the composite electrospun nanofibrous mats containing triethylene glycol diacrylate as cross-linking agent had resulted in stabilizing of the nanofibers against disintegration in water or water vapors.Ignatova et al. (2007)also prepared QCS-containing nanofibers by electrospinning with PVP blending. A significant decrease in the fiber diameter of electrospun QCS/PVP mixed fibers and a narrowing of thefiber diameter distribution with increasing the QCS content was observed and explained by the increase in solution conductivity. An increase of the appliedfield strength led to greater fiber diameters and to broader diameter distribution.

The galactosylated chitosan (GC) nanofibrous scaffold with an average diameter of∼160 nm was fabricated by electrospinning using formic acid for tissue engineering applications (Feng et al., 2009).Jiang et al. (2004)prepared electrospun membranes composed of ibuprofen-loaded poly(lactide-co-glycolide)/poly(ethylene glycol)-g-chitosan (PLGA)/(PEG-g-chitosan). Due to their high porosity, these membranes could be suitable for a trialfibrillation (Reneker and Chun, 1996). In this work, ibuprofen was incorporated into thefibrous mats in two different methods: electrostatically conjugated during the electrospinning process and covalently conjugated to the PEG-g- chitosan prior to spinning. This tri-component system had unique properties of being soluble in organic solvents, while being insoluble in neutral pH water (Ouchi et al., 1998). It was also demonstrated that the hydrophilicity, membrane shrinkage, and rate of drug release could be controlled within the system. Thefibrous mats in vitro and in vivo biocompatibility and efficacy need to be evaluated.Duan et al. (2006;

2007)simultaneously electrospun PLGA and chitosan/PVA onto a rotating drum using two different syringes. The PLGA component in the composites was expected to enhance the mechanical properties, while the chitosan provides bioactivity and biocompatibility. The ﬁbrous mats were cross-linked with 25% aqueous glutaraldehyde vapor for 4 h at 37 °C (Chen et al., 2003). It was determined that tri- component systems might have potential in biomedical applications.

Wan et al. (2008)prepared poly(chitosan-g-DL-lactic acid) (PCLA) copolymeric nanoﬁbers using an electro-wet-spinning technique. The diameter ofﬁbers in different scaffolds was found to be about 100 nm.

Two main processing parameters, that is, the concentration of PCLA solutions and the composition proportions of coagulation solutions, were optimized for obtaining desired scaffolds with well-controlled structures. The tensile properties of the scaffolds in both dry and hydrated states were examined. Signiﬁcantly improved tensile strength and modulus for theseﬁbrous scaffolds in their hydrated

state were observed.Table 1summarizes the experimental conditions adapted for the fabrication and obtained average size of electrospun chitosan nanoﬁbers.

3. Applications of chitin and chitosan nanoﬁbers 3.1. Filtration

Chitosan based nanofibers have desired filtration properties when compared to nanofibers fabricated from other synthetic polymers. It was used in a wide variety offiltration applications ranging from water purification media to air filter media. Desai et al. (2009) fabricated nanofibrous filter media by electrospinning of chitosan/

PEO blend solutions onto a spunbonded non-woven polypropylene substrate. They demonstrated the usage of chitosan based nanofibrous filter media to effectively filter out heavy metal ions, pathogenic micro-organisms, and contaminant particulate media from both air and water media. Heavy metal binding, anti-microbial and physical filtrations efficiencies of these chitosan based filter media were studied and correlated with the surface chemistry and physical characteristics of these nanofibrous filter media. Filtration efficiency of the nanofiber mats was strongly related to the size of the fibers and its surface chitosan content. Hexavalent chromium binding capacities up to 35 mg chromium/g chitosan were exhibited by chitosan based nanofibrous filter media along with a 2–3 log reduction in E. coli bacteria. After 6 h of contact time the chitosan blendfibers did show a 2–3 log reduction in E. coli. Air and water filtration efficiencies of the nanofibrous filter media measured using aerosol and PS beads suspended in water respectively showed high efficiencies which correlated with thefibrous media size and shape. These results indicated the advantage of chitosan nanofibers in filters and its commercial applicability.

The metal adsorbability of the chitosan electrospun nanofiber (∼235 nm in diameter) mats was examined in an aqueous solution (Haider and Park, 2009). The chitosan nanofiber mats neutralized with potassium carbonate showed good erosion stability in water and high adsorption affinity for metal ions in an aqueous solution. The adsorption data of Cu(II) and Pb(II) werefitted well with Langmuir isotherm indicating that mono-layer adsorption occurred on the nanofiber mats. The equilibrium adsorption capacities for Cu(II) and Pb(II) were 485.44 mg g^{− 1}and 263.15 mg g^{− 1}, respectively. The Cu (II) adsorption data were∼6 and ∼11 times higher than the reported highest values of chitosan microsphere (80.71 mg g^{− 1}) (Wan Ngah et al., 2002), and plain chitosan (45.20 mg g^{− 1}) (Huang et al., 1996), respectively. This high adsorption capacity suggests that the chitosan electrospun nanofiber mats can be applied to filter out (or neutralize) toxic metal ions and microbes without losing their original chitosan properties such as biocompatibility, hydrophilicity, bioactivity, non- antigenicity and non-toxicity.

3.2. Biosensors

Nanofibrous chitosan/PVA membrane was developed for enzyme immobilization (Huang et al., 2007). This chitosan nanofibrous membrane was used as a support for lipase immobilization with the advantages of high enzyme loading up to 63.6 mg g^{− 1}and activity retention of 49.8%. The stabilities of the immobilized lipase towards pH, temperature, reuse and storage were enhanced. These results imply that the chitosan nanofibrous membrane with excellent biocompatible is a potential support for enzyme immobilization. This system can be used for biosensor applications.

3.3. Wound dressing

It is known that chitosan derivatives with quaternary ammonium groups possess high efﬁcacy against bacteria and fungi. It is now

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widely accepted that the target site of these cationic polymers is the cytoplasmic membrane of bacterial cells (Tashiro, 2001). Micro- and nanofibrous materials are suitable for preparing wound dressings. The photo-cross-linked electrospun mats containing QCS were efficient in inhibiting growth of Gram-positive bacteria and Gram-negative bacteria (Ignatova et al., 2007). These results suggested that the cross-linked QCS/PVP electrospun mats are promising materials for wound-dressing applications. Similarly, the photo-cross-linked electrospun nanofibrous QCS/PVA mats had a good bactericidal activity against the Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus (Ignatova et al., 2006). These characteristic features of the electrospun mats reveal their high potential for wound-dressing applications. The best biomaterials for wound dressing should be biocompatible and promote the growth of dermis and epidermis layers. Chen et al. (2008a,b,c) reported composite nanofibrous membrane of chitosan/collagen, which are known for their beneficial effects on wound healing. The membrane was found to promote wound healing and induce cell migration and proliferation. From animal studies, the nanofibrous membrane was found to be better than gauze and commercial collagen sponge in wound healing.

3.4. Tissue engineering

Polymeric nanoﬁbers that mimic the structure and function of the natural extracellular matrix (ECM) are of great interest in tissue engineering as scaffolding materials to restore, maintain or improve the function of human tissues. The natural ECMs in the body are mainly composed of two classes of extracellular macromolecules:

proteoglycans andﬁbrous proteins with ﬁber diameters ranging from 50 to 150 nm, depending on tissue type (Elsdale and Bard, 1972).

Recent studies showed that the material size feature could substan- tially inﬂuence the morphology and function of cells grown on the ECM. The cells attachment and proliferation were found to be good on micro and nanostructured materials (Laurencin et al., 1999; Teixeira et al., 2003).

Collagen is a major natural extracellular matrix component, and possesses afibrous structure with fiber bundles varying in diameter from 50 to 500 nm (Hay, 1991; Elsdale and Bard, 1972). Many efforts have been made tofind an alternative scaffold material with similar physicochemical and biological characteristics of ECM (Chiu et al., 2007). The morphology of electrospun nanofiber mat is very similar to the morphology of human native ECM (Zhang et al., 2005a,b;

Venugopal et al., 2008), thus electrospun nanofiber could be a promising scaffolding material for cell culture and tissue engineering application. The electrospinning process makes it possible to produce complex, seamless and three-dimensional (3D) nanofiber scaffolds that support diverse types of cells to grow into the artificial tissues.

Noh et al., 2006studied the cytocompatibility of chitin nanoﬁbers.

Chitin nanofibers were found to promote cell attachment and spreading of normal human keratinocytes andfibroblasts compared to chitin microfibers. This may be a consequence of the high surface area available for cell attachment due to their three-dimensional features and high surface area to volume ratios, which are favorable parameters for cell attachment, growth, and proliferation. Cell studies conducted on chitin/PGA (Park et al., 2006a) and chitin/SF (Park et al., 2006b)fibrous mats proved that a matrix consisting of 25% PGA or SF and 75% chitin had the best results. The chitin/PGAfibers had a bovine serum albumin coating and were considered as a good candidate for use as a tissue engineering scaffold. The chitin/SFfibrous mats had the highest spreading of normal human epidermalfibroblasts (NHEF) and normal human epidermal keratinocytes (NHEK). Therefore, these scaffolds were suggested for wound tissue engineering applications.

Shalumon et al. (2009) developed CMC/PVA blend nanofibrous scaffold for tissue engineering applications. The prepared nanofibers were found to be bioactive and biocompatible. Cytotoxicity and cell attachement studies of the nanofibrous scaffold were evaluated using

human mesenchymal stem cells (hMSCs) by the MTT assay. The cell attachment studies revealed that cells were able to attach and spread on the nanoﬁbrous scaffolds (Fig. 1). These results indicate that the nanoﬁbrous CMC/PVA scaffold supports cell adhesion/attachment and proliferation and hence these scaffolds are useful for tissue engineering applications.

Fig. 1. SEM images of hMSCs attached on the surfaces of CMC/PVA scaffolds after (a) 12 h (b) 24 h and (c) 48 h of incubation.

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Bhattarai et al. (2005)reported that the chitosan/PEO nanofibrous scaffolds promoted the attachment of human osteoblasts and chondrocytes and maintained characteristic cell morphology and viability throughout the period of study. This nanofibrous matrix is of particular interest in tissue engineering for controlled drug release and tissue remodeling. Similarly,Subramanian et al. (2005)prepared chitosan/PEO nanofibers for cartilage tissue engineering. Chitosan/

PEO nanofibers showed the biocompatibility with chondrocytes. Cells were attached to the chitosan/PEO nanofiber mats and the results indicated that the electrospun chitosan/PEO mats could be used for cartilage tissue repair.Mo et al. (2007)also reported the smooth muscle cells attached to the electrospun chitosan/collagen nanofibers for tissue engineering applications.

The biological evaluations of chitosan/HAp nanﬁbrous composite scaffolds have been reported (Zhang et al., 2008a,b). The chitosan/

HAp nanofibrous scaffolds have significantly stimulated the bone forming ability due to the excellent osteoconductivity of HAp compared to the control chitosan. The results obtained from this study highlight the great potential of using the chitosan/HAp nanocomposite nanofibers for bone tissue engineering applications. A biocomposite nanofibrous scaffolds were prepared by using chitosan/

PVA and N-CECS/PVA (Fig. 2) for tissue engineering applications (Yang et al., 2008). The cell attachment of the prepared biocomposite nanofibers was studied using mouse fibroblast (L929) cell. The L929 cell culture revealed the attachment and growth of mousefibroblast on the surface of biocomposite scaffolds. Similarly, the potential use of the N-CECS/PVA electrospunfiber mats as scaffolding materials for skin regeneration was evaluated in vitro using L929 (Zhou et al., 2008). Indirect cytotoxicity assessment of thefiber mats indicated that the N-CECS/PVA electrospun mat was non-toxic to the L929 cell.

Cell culture results showed thatﬁbrous mats were good in promoting

the cell attachment and proliferation. This electrospun matrix would be used as potential wound dressing for skin regeneration.

Liver tissue engineering requires a perfect extracellular matrix (ECM) for primary hepatocytes culture to maintain high level of liver- speciﬁc functions and desirable mechanical stability.Feng et al. (2009) developed GC nanoﬁbers with surface-galactose ligands to enhance the bioactivity and mechanical stability of primary hepatocytes in culture.

The GC nanofibrous scaffolds displayed slow degradation and suitable mechanical properties as an ECM for hepatocytes according to the evaluation of disintegration and Young's modulus testing. The hepatocytes cultured on GC nanofibrous scaffold formed stable immobilized 3D flat aggregates and exhibited superior cell bioactivity with higher levels of liver-specific function maintenance in terms of albumin secretion, urea synthesis and cytochrome P-450 enzyme than 3D spheroid aggregates formed on GCfilms. These results suggested that the GC- based nanofibrous scaffolds could be useful for various applications such as bioartificial liver-assist devices and tissue engineering for liver regeneration as primary hepatocytes culture substrates.

3.5. Drug delivery

Electrospun nanofibers have been used for drug delivery applications. The drug loading was found to be very simple to implement via electrospinning process, and the high-applied voltage used in the electrospinning process had little influence on the drug activity. The high specific surface area and short diffusion passage length give the nanofiber drug system higher overall release rate than the bulk material.

The release profile can be finely controlled by modulation of nanofiber morphology, porosity and composition. Nanofibers for drug release systems mainly come from the biodegradable polymers such as PVA, PLA, PEO, PCL, PEG, PLGA and chitosan.Jiang et al. (2004)developed ibuprofen drug loaded electrospun PEG-g-chitosan with PLGA for controlled drug delivery applications. The presence of PEG-g-chitosan significantly moderated the burst release rate of ibuprofen from the electrospun PLGA membranes. Moreover, in this study, ibuprofen was conjugated to the side chains of PEG-g-chitosan for the prolonged release for more than 2 weeks (Fig. 3). These results indicated that chitosan nanofibers could be useful for controlled drug delivery applications.

4. Conclusions

This review summarized the preparation and biomedical applications of nanofibers based on chitin, chitosan and their derivatives in thefields of tissue engineering, wound healing, biosensors, drug Fig. 2. SEM images of (a) and (b) L929 cells seeded onfibrous membranes of HAp-N-

CECS/PVA after 48 h culture.

Fig. 3. Release proﬁles of ibuprofen from an electrospun (A) PLGA membrane (5%

ibuprofen), (B) a PLGA/PEG-g-chitosan membrane (5% ibuprofen), and (C) a PLGA/PEG- g-chitosan membrane conjugated with ibuprofen (4.4% ibuprofen). Electrospun membranes were incubated in 0.1 M PBS (pH 7.4) at 37 °C.

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release andﬁltration process. From this review, it is concluded that chitin and chitosan based nanoﬁbers have the potential importance for the development of conventional and novel pharmaceutical products.

Because of their favorable biological properties such as non-toxicity, biocompatibility, biodegradability, and antibacterial activity, these nanofibers are promising candidates for the enhancement of absorp- tion of drugs, enzyme immobilization, cell proliferation and wound healing. However, most of these applications are still at the laboratory level. Additional studies are necessary before we can expect clinical applications and commercialization of chitin and chitosan based nanofibers. We hope that this review will help promote new innovative types of chitin and chitosan based nanofibers for biomedical application in the near future.

Acknowledgments

One of the authors R. Jayakumar is grateful to SERC Division, Department of Science and Technology (DST), India, for providing the fund under the scheme of“Fast Track Scheme for Young Investigators”

(Ref. No. SR/FT/CS-005/2008). Dr. S. V. Nair is also grateful to DST, India, which partially supported this work, under a center grant of the Nanoscience and Nanotechnology Initiative program monitored by Dr. C. N. R. Rao. The author R. Jayakumar is also grateful to Department of Biotechnology (DBT), Govt. of India for providing research support.

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