Review
Chitosan based nano fibers, review
Maher Z. Elsabee
a,⁎ , Hala F. Naguib
a, Rania Elsayed Morsi
baDepartment of Chemistry, Faculty of Science, Cairo University, Cairo, 12613, Egypt
bEgyptian Petroleum Research Institute, Nasr City, Cairo, 11727, Egypt
a b s t r a c t a r t i c l e i n f o
Article history:
Received 29 September 2011 Received in revised form 12 March 2012 Accepted 9 May 2012
Available online xxxx
Keywords:
Chitin Chitosan Electrospinning Chemical modifications Blends, nano-fibers
Chitin and chitosan are natural polymers with a huge potential in numerousfields, namely, biomedical, bio- logical, and many industrial applications such as waste water treatment due to the fact that they can absorb and chelate many metal cations. Electrospinning is a growingfield of research to produce submicron fibers with promising applications in biomedicalfields like tissue engineering scaffolds and wound healing capabil- ities. Both chitin and chitosan polymers were found to be hard to electrospun, however, many researchers manage to produce nano-fibers using special solvents; for example, 90% acetic acid was found to reduce the surface tension making electrospinning feasible. Mixtures of organic acids were also experimented to produce homogenous and uniform fibers. Bigger attention was given to electrospinning of their soluble derivatives such as dibutyryl and carboxymethyl chitin. More derivatives of chitosan were investigated to produce nano-fibers such as hexanoyl, polyethyleneglycol, carboxymethyl, and a series of quaternized chito- san derivatives. The obtained nano-fibers were found to have much better qualities than normal chitosan fibers. Several polymer blends of chitin/chitosan with many commercial polymers were found to be amena- ble for electrospinning producing uniform beads freefibers. The review surveys the various approaches for successful electrospinning of chitin, chitosan, their derivatives, and blends with several other polymers.
© 2012 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . 0
1.1. Electrospinning process . . . 0
1.2. Factors affecting the electrospinning process . . . 0
1.3. Electrospinning of biodegradable polymers . . . 0
2. Chitin and chitosan electrospinning . . . 0
2.1. Electrospinning of chitin . . . 0
2.2. Electrospinning of chitin derivatives and blends . . . 0
2.2.1. Electrospinning of dibutyryl chitin . . . 0
2.2.2. Electrospinning of poly (vinyl alcohol) (PVA) containingα-chitin whiskers . . . 0
2.2.3. Carboxymethyl chitin (CMC)/poly (vinyl alcohol) (PVA) blend . . . 0
2.2.4. Chitin/poly (glycolic acid) (PGA) . . . 0
2.2.5. Chitin/silkfibroin (SF). . . 0
2.2.6. Electrospinning of dibutyryl chitin (DBC) and cellulose acetate (CA) blends . . . 0 Materials Science and Engineering C xxx (2012) xxx–xxx
Abbreviations: HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; Chi-N, chitin nanofibrous matrix; Chi-M, chitin microfibers; (Ce), entanglement concentration; cP, centi Poise; DBC, dibutyryl chitin; PVA, Poly (vinyl alcohol); NHEK, Normal Human Epidermal Keratinocytes; NHEF, Normal Human Fibroblasts; CMC, Carboxymethyl chitin; PGA, poly (glycolic acid); PBS, phosphate-buffered saline; SF, silkfibroin; CA, cellulose acetate; FA, formic acid; DCA, dichloroacetic acid; TFA, trifluoroacetic acid; DCM, dichloromethane; GA, glutar- aldehyde; H-chitosan, hexanoyl chitosan; PF, pyridinum formate; PEG, poly (ethylene glycol); CMC, carboxymethyl chitosan; DS, degree of substitution; PEO, poly(ethylene oxide);
PAAM, polyacrylamide; PAA, poly(acrylic acid); CS, chitosan; PEGDMA, polyethyleneglycol-600-dimethylacrylate; HEPK, 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1- propanone; COS, chitosan oligosaccharide; MMT, montmorillonite clay; SDS, anionic sodium dodecyl sulfate; Brij 35, nonionic polyoxyethylene glycol lauryl ether; DTAB, cationic dodecyl trimethyl ammonium bromide; TFA, trifluoroacetic acid; DCM, dichloromethane; PAAm, poly acrylamide; PLA, polylactides; Ch/PLA, chitosan/polylactides; QCh/PLA, quaternized Chitosan/polylactides; TEGDA, triethylene glycol diacrylate; DMPA, 2,2-dimethoxy-2-phenylacetophenone; HTCC, N-[(2-hydroxy-3-trimethylammonium) propyl]chi- tosan chloride; PVP, poly(vinyl pyrrolidone); AFS, appliedfield strength; MIC, Minimum inhibitory concentration; PLGA, poly lactide-co-glycolide; THF, tetrahydrofuran; DMF, N,N- dimethlformamide; GAGs, glycosamino-glycans; ECM, electrospun composite membranes; Ag NPs, silver nanoparticles; CEC, carboxyethylchitosan; SEM, scanning electron micros- copy; EDX, energy dispersive X-ray; XPS, X-ray photoelectron spectroscopy; S. aureus, Staphylococcus aureus; E. coli, Escherichia coli.
⁎ Corresponding author. Tel.: +20 106680474 (mobile).
E-mail address:mzelsabee@yahoo.com(M.Z. Elsabee).
MSC-03426; No of Pages 16
0928-4931/$– see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2012.05.009
Contents lists available atSciVerse ScienceDirect
Materials Science and Engineering C
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m s e c
3. Electrospinning of chitosan . . . 0
3.1. Electrospinning of chitosan derivatives . . . 0
3.1.1. Hexanoyl chitosan . . . 0
3.1.2. PEGylation of chitosan . . . 0
3.1.3. Carboxymethyl chitosan (CMC). . . 0
3.2. Electrospinning of chitosan blends . . . 0
3.2.1. Chitosan-poly (vinyl alcohol) blend . . . 0
3.2.2. Crosslinked chitosan/PVA blend . . . 0
3.2.3. Chitosan–polyethylene oxide blends . . . 0
3.2.4. Collagen–chitosan blend . . . 0
3.2.5. Chitosan–agarose blend . . . 0
3.2.6. Chitosan/polyacrylamide . . . 0
3.2.7. Chitosan/zein blend . . . 0
3.2.8. Nylon-6/chitosan blends . . . 0
4. Quaternized chitosan blends. . . 0
4.1. Quaternized chitosan/polylactides . . . 0
4.2. Quaternized chitosan/poly (vinyl alcohol) . . . 0
4.3. Quaternized chitosan/poly(vinyl pyrrolidone) . . . 0
5. Poly lactide-co-glycolide (PLGA)/chitosan/poly (vinyl alcohol) (PVA). . . 0
6. Electrospinning of chitosan derivatives and silver nanoparticles . . . 0
7. Conclusion . . . 0
References . . . 0
1. Introduction
1.1. Electrospinning process
Electrospinning has been recognized as an efficient technique for the fabrication of submicron-sized polymer nanofibers ranging from 5 to 500 nm, 102to 104times smaller than those prepared by the tra- ditional methods of solution or melt spinning[1]. Various macromol- ecules have been successfully electrospun into ultrafine fibers as thin as several nanometers. In terms of the flexibility of the process, electrospinning is able to fabricate continuous nanofibers from a huge range of materials.
Electrospinning has attractive applications since it has a potential to fabricate natural tissue in vitro with diameter in the nano-scale range close to the collagenfibers found in the natural extracellular matrix [2]. Nanofibers have amazing characteristics such as very large surface area-to-volume ratio and high porosity with very small pore size. Therefore, nanofibers can be promising materials for many biomedical applications[3,4]such as tissue templates, medical prostheses, artificial organ, wound dressing, bone tissue engineering [5], drug delivery[6], pharmaceutical composition[7–9],filter forma- tion and sensing applications[10,11].
The formation of nanofibers through electrospinning is based on the uniaxial stretching of a viscoelastic solution. There are basically three components in an electrospun setup: a high voltage power sup- ply, a capillary tube with a needle and a metal collector[12].
In an electrospinning process, an electricfield is applied between the needle capillary end and the collector so that surface charge is in- duced on a polymerfluid deforming a spherical pendant droplet to a conical shape[13]. As the electricfield surpasses a threshold value where electrostatic repulsion force of the surface charges overcome surface tension, the chargedfluid jet is ejected from the tip of the con- ical protrusion commonly referred to as Taylor cone[14]and the charge density on the jet interacts with the externalfield to produce instability. High surface charge densities enhance a whipping mode rather than axisymmetric mode, where bending of the jet produces highly stretched polymericfiber with simultaneous rapid evaporation of the solvent.
The main advantage of the electrospinning nano-manufacturing process is that it is cost effective compared to that of most bottom– up methods. The nanofibers prepared from electrospinning process are often uniform and continuous and do not require expensive
purification unlike submicrometer diameter whiskers, inorganic nanorods, and composite material reinforcement.
1.2. Factors affecting the electrospinning process
Important parameters in electrospinning are not only polymer and solution properties such as molecular weight, viscosity, conduc- tivity and surface tension, but also electrospinning conditions such as applied electric voltage, tip-to collector distance, feeding rate, etc [15,16]. A critical concentration is needed in order to obtainfibers from electrospinning. Below this concentration, chain entanglements are insufficient to stabilize the Coulombic repulsion within the ejected jet, leading to the formation of sprayed droplets. When the applied electricfield can overcome the surface tension of the polymer solution, uniform and homogeneousfibers were formed. Generally, surface tension determines the upper and lower boundaries of electrospinning window if all other variables are held constant. The formation of droplets, bead andfibers can be driven by the surface tension of polymer solution; lowering the surface tension of solution helps electrospinning to occur at lower electricfields[17,18].
1.3. Electrospinning of biodegradable polymers
For many biomedical applications, the most important character- istics that should be targeted include biocompatibility and mechani- cal performance. In comparison with synthetic counterparts, natural biopolymers generally have better biocompatibility and hence are more suitable for human body. However, to convert a natural bio- polymer into submicron or nanometerfibers through electrospinning is usually more difficult than to do a synthetic polymer[19]. Due to this reason, relatively few reports addressing electrospinning of some natural biopolymers can be found in the literature[20–29]. 2. Chitin and chitosan electrospinning
Chitosan has proven challenging to electrospin. First, chitosan rigidD-glucosamine repeat unit, high crystallinity and ability to hy- drogen bond lead to poor solubility in common organic solvents.
McKee et al.[30]found that in order to electrospin defect-freefibers from polymer solutions, the concentration of the polymer must be at least 2 to 2.5 times the entanglement concentration. The entangle- ment concentration (Ce) is the boundary between the semi dilute un-
entangled regime (where polymer chains overlap one another but are not entangled) and the semi dilute entangled regime (where the polymer chains significantly overlap one another and topologically constrain each other's motion). However, chitosan solutions at these concentrations are often difficult to electrospin because of the high viscosity in solution. With chitosan solutions, even moderate concen- trations become too viscous to overcome the electricfield and cannot be successfully electrospun. Additionally, chitosan is a cationic bio- polymer that also affects the rheology of the solutions. With a typical laboratory electrospinning setup, the viscosity of the solution must be within a certain window for nanofibers to form successfully. Above the upper threshold, the solution becomes too viscous andfiber for- mation is hindered because the electricfield is not strong enough to overcome the viscosity of the solution. Below the lower limit, the polymer chains are not entangled sofiber formation is not possible and polymer beads often are created. Therefore, several research groups have blended chitosan with other polymers in an attempt to improve the electrospinability of the solutions[31].
2.1. Electrospinning of chitin
Despite its limited solubility, nonwoven wound dressing nano- fibers of chitin were fabricated via electrospinning in 1,1,1,3,3,3- hexafluoro-2-propanol (HFIP)[32]. The limited solubility of chitin in HFIP was enhanced by degrading chitin withγ-irradiation to reduce its molecular weight. A high electric potential (15 kV) was applied to a droplet of chitin solution at the tip (0.495 mm in internal diame- ter) of a syringe needle. Concentrations of chitin solutions were in the range from 1 to 6% by weight. The electrospun chitin nanofibers were collected on a target drum which was placed at a distance of ~7 cm from the syringe tip.
SEM micrographs of the formed nanofibers electrospun are shown inFig. 1. At the concentration up to 3% by weight, large irregular beads or beadedfibers were generated by electrospinning. The con- tinuous nanofibers can be obtained at the concentration above 4%
by weight, and this concentration appears to correspond to the onset of significant chain entanglements in the solution. At 6 wt.%
concentration, the continuous and uniform electrospinning was inhibited because chitin solution had very high viscosity (5310 cP).
The resultantfibrous structure containing irregular, but small, beads was observed again. The as-spun nanofibrous chitin matrix consists offibers with average diameter of 110 nm.
Noh et al.[33]also fabricated chitin nanofibrous matrix (Chi-N) by electrospinning in HFIP to be used as a biodegradable wound dressing or scaffold for tissue engineering. The concentrations of the chitin so- lutions used for electrospinning were in the range of 3–6% (by weight). In the electrospinning process, a high electric potential (17 kV) was applied to a droplet of chitin solution at the tip (0.55 mm in internal diameter) of a syringe needle.
SEM micrographs of the fabricated chitin nanofibrous matrix (Chi- N) and the commercial chitin microfibers (Chi-M) show that the Chi- N matrix has an average diameter of 163 nm (with diameters ranging from 50 to 460 nm), while the average diameter of the Chi-M was 8.77μm.
Among the remarkable characteristics of the electrospunfibers is the higher rate of degradation and the authors attributed this en- hanced degradation rate to the lower molecular weight of thefibers rather than to their smaller diameters. The results showed that Chi- N is degraded well, starting 14 days after implantation in vivo, and anticipating that the chitin nanofibrous matrices may be suitable for biomedical applications, such as tissue regeneration and wound dressings.
Moreover, both types of the chitin matrix surfaces were modified using type I collagen,fibronectin or laminin, which were adsorbed onto the chitin matrices as substrates. Relatively high cell attachment and spreading of all the cells tested were observed on Chi-N in com- parison to Chi-M, and Chi-N treated with type I collagen that signifi- cantly promoted the cellular response. These results indicate that the Chi-N, alone or with extracellular matrix proteins (particularly type I collagen), could be potential candidates for the cell attachment and spreading of normal human keratinocytes andfibroblasts. This property of Chi-N might be particularly useful for wound healing and regeneration of oral mucosa and skin.
2.2. Electrospinning of chitin derivatives and blends
2.2.1. Electrospinning of dibutyryl chitin[34]
Due to the difficulty of dealing with chitin and the limitation of its solubility, the attention was diverted to the electrospinning of its sol- uble derivatives, as a promising candidate was dibutyryl chitin.
Dibutyryl chitin (DBC), the organic soluble chitin derivative, has been successfully processed intofibers via conventional dry spinning from acetone producing dumb-bell cross sectionalfibers that were 11–23 μm wide and 40–120 μm long [35], whereas wet spinning
Fig. 1. SEM micrographs of chitin nanofibers electrospun at different concentrations[32].
M.Z. Elsabee et al. / Materials Science and Engineering C xxx (2012) xxx–xxx 3
from DMF produced more uniform and nearly circularfibers with 20μm diameters[36,37]. Muchfiner DBC fibers (300 nm diameter) have been electrospun from ethanol[38]. The dibutyryl chitin nano- fibers were then treated with alkali solution to regenerate chitin fi- bers. The dibutyryl chitin fibers have been used to prepare nonwoven materials with good wound healing property[39].
2.2.2. Electrospinning of poly (vinyl alcohol) (PVA) containingα-chitin whiskers
Junkasem et al.[40] fabricated nanocompositefiber mats com- posed of electrospun poly (vinyl alcohol)s (PVA) containingα-chitin whiskers. This work was conducted since PVA had been electrospun in water[41] and chitin/PVA, nanocomposite films containing α- chitin whiskers have also been fabricated [42]. It was found that blending increases the tensile strength; the maximum tensile strength value of 5.7 ± 0.6 MPa was obtained when the chitin whisker to PVA ratio was approximately 5.1%. Beyond this ratio point, increasing the chitin content decreased the strength of the mats[40].
2.2.3. Carboxymethyl chitin (CMC)/poly (vinyl alcohol) (PVA) blend Shalumon et al.[43]developed a novel electrospun water-soluble carboxymethyl chitin (CMC)/PVA blend. The concentration of CMC (7%) with PVA (8%) was optimized, blended in different ratios (0–100%), and electrospun to get nanofibers. Fibers were made water insoluble by cross-linking with glutaraldehyde vapors followed by thermal treatment. In vitro mineralization studies identified the ability of formation of hydroxyapatite deposits on the nanofibrous surfaces. Cytotoxicity of the nanofibrous scaffold was evaluated. SEM images revealed that cells were able to attach and spread in the nanofibrous scaffolds. Thus, this nanofibrous CMC/PVA scaffold sup- ports cell adhesion/attachment and proliferation and hence this scaf- fold will be a promising candidate for tissue engineering applications.
2.2.4. Chitin/poly (glycolic acid) (PGA)
Park et al.[44]developed electrospun chitin/poly (glycolic acid) (PGA) blend nanofibers in HFIP. PGA was chosen because it was a bio- compatible and biodegradable polymer. The PGA/chitin blendfibers have average diameters of around 140 nm, and their diameters have a distribution in the range 50–350 nm. In vitro degradation studies, conducted in phosphate-buffered saline (PBS) pH 7.2, demonstrated that the blend fibers degraded faster than pure PGA fibers. This blend can be used to fabricate biodegradable and biomimetic nano- structured scaffolds for tissue engineering.
2.2.5. Chitin/silkfibroin (SF)
An electrospun chitin/silkfibroin (SF) nanofiber in HFIP solvent was reported[45]. SF was extensively studied as one of the candidate materials for biomedical applications, because it has several distinc- tive biological properties including good biocompatibility, biodegrad- ability, and minimal inflammatory reaction. The average diameters of chitin/SF blendfibers decreased from 920 to 340 nm with the in- crease of chitin content in blend compositions. The cyto- compatibility and cell behavior on the chitin/SF blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal keratinocyte andfibroblasts seeded on the scaffolds were studied. The results indicate that chitin/SF blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds because it has both biomi- metic three-dimensional structure and an excellent cell attachment and spreading for normal human epidermal keratinocytes (NHEK) andfibroblasts (NHEF)[45].
2.2.6. Electrospinning of dibutyryl chitin (DBC) and cellulose acetate (CA) blends[34]
Electrospinning mixtures of ester derivatives of cellulose and chi- tin followed by aqueous hydrolysis have proven to be a facile
approach to generate cellulose–chitin and cellulose–chitosan hybrid nanofibers. The readily available cellulose derivative (CA), the easily derived chitin derivative (DBC) and the highly compatible nature of these derivatives allow their full integration to generate complete gradient of cellulose–chitosan hybrid compositions. The single-step, simultaneous hydrolysis of CA and DBC is advantageous and highly efficient to regenerate the final cellulose–chitosan nanofibrous products.
Electrospinning of CA and DBC mixtures from acetone, DMAc, acetic acid, and their mixtures showed the optimal solvent system for the CA/DBC hybrid systems to be 1/1 w/w acetone/acetic acid mix- ture. At afixed 15% in 1/1 w/w acetone/acetic acid mixture, CA and DBC with varies compositions from 100/0 to 0/100 ratios led to uni- form nanofibers with 30–350 nm diameters. Both FT-IR and DSC data support the absence of phase separation between CA and DBC in the hybrid nanofibers.
Aqueous alkaline hydrolysis from 0.5 to 10 N NaOH at ambient temperature for 24 h or 100 °C for 3 h, readily regenerated more than 95% of cellulose and chitin from CA and DBC, irrespective of the conditions. The conversion of C-2 acetyl amine to primary amino groups, or the deacetylation of chitin to chitosan was, howev- er, more favored at either the higher NaOH concentrations (5 and 10 N) or the higher temperature (100 °C). A 90.6% deacetylated chito- san was achieved with the 10 N NaOH at 100 °C for 3 h, although much of thefibrous structure was lost. The optimal hydrolysis condi- tion to retain thefiber morphology was with 5 N NaOH at 100 °C for 3 h, yielding chitosan at 81.3% deacetylation.
3. Electrospinning of chitosan
Chitosan was electrospun in several solvent systems across a broad range of concentrations; however, no ultrafine fibers were obtained even at the concentrations in which the chitosan chains were extensively entangled[4]. Therefore, the work was directed to obtain chitosan nanofibrous matrix from the neutral nonionic form of chitin through the deacetylation of electrospun chitin matrix. Chi- tin nanofibers were regenerated into chitosan nanofibers via hetero- geneous deacetylation using 40% by wt NaOH at a high temperature (100 °C), the degree of deacetylation rapidly increased up to 85%
within 2 h[32]. This result indicates that chitin in the nanofibers form deacetylates much faster than in the usual powder form under these conditions. The structural transformation from chitin to chito- san was confirmed by FTIR and WXRD. SEM micrographs and the corresponding diameter distributions of chitin and deacetylated chi- tin, chitosan, nanofibers (Fig. 2) showed no significant morphological changes.
In another study, chitosan nanofibers were electrospun from aqueous chitosan solution using concentrated acetic acid solution as a solvent[13]. At acetic acid concentration of 30% or more, several thin nanofibers about 40 nm in average diameter were initially pro- duced together with large beads and at 90% acid concentration thefi- bers diameter increased to 130 nm without beads formation. A uniform nanofibrous mat of average fiber diameter of 130 nm was obtained by electrospun 7% chitosan solution in aqueous 90% acetic acid in 4 kV/cm electricfield. One of the most interesting results from these studies[13]is that the acetic acid concentration in water strongly influenced surface tension of chitosan solutions, which was remarkably important in chitosan electrospinning. As acetic acid con- centration increased from 10% to 90%, surface tension decreased from 54.6 dyn/cm to 31.5 dyn/cm without significant viscosity change. Net charge density of the chitosan solution also increased with increasing acetic acid concentration in water resulting in more charged ions available for charge repulsion[13].
Among three used chitosan samples with different molecular weights and concentrations, only about 7–7.5% of 106,000 g/mol chi- tosan (viscosity ranging from 484 to 590 cP) can produce a
continuous and uniformfiber. The electrospun samples of the lower molecular weight chitosan solution (9.5–10.5%) usually contained large size beads and fragilefibers, while those of the higher molecular weight chitosan solution (2.5–3%) showed rougher and finer nano- fibers with some bead defects.
Ohkawa et al.[46]also investigated the effect of chitosan concen- tration and solvents on the chitosan electrospinning ability. Among the solvents tested were dilute acetic and hydrochloric acids, neat formic acid FA, dichloroacetic acid DCA and trifluoroacetic acid TFA.
For example, the morphology of the deposited chitosan was found to be depended on its concentration in the TFA solution. When the chitosan concentration was 6 wt.% or less, the beads and fibers coexisted in the SEM images. At a concentration of 7 wt.%, fibers were predominantly deposited while the bead fraction remarkably decreased (average diameter, 490 nm; diameter distribution, 330–610 nm). An almost homogenous network of the electrospun chitosan fibers was observed at 8 wt.% with average diameter, 490 nm; diameter distribution, 390–610 nm.
Successful electrospinning of chitosan in TFA as a solvent was at- tributed by the authors[46]to: (i) TFA forms salts with the amino groups of chitosan and this salt formation destroys the rigid interac- tion between the chitosan molecules, making them ready to be electrospun; which is unconvincing since most acids form salts with chitosan (ii) the high volatility of TFA is advantageous for the rapid solidification of the electrified jet of the chitosan–TFA solution.
To further optimize the electrospinning process another volatile solvent, dichloromethane (DCM), was added in different ratios and the electrospinning process was conducted. The best conditions for the production of almost beads freefibers were when the solvent ratio was 70:30 TFA:DCM. The mean diameter of the chitosanfiber thus obtained was 330 nm and thefiber diameters were distributed from 210 to 650 nm.
Schiffman et al.[47]have also successfully electrospun unfiltered low, medium and high molecular weight chitosan, as well as practical-grade chitosan into nanofibrous mats. The as-spun medium molecular weight chitosan nanofibers have a Young's modulus of 154.9 ± 40.0 MPa and display a pseudo-yield point that arose due to the transition from the pulling of afibrous mat with high cohesive strength to the sliding and elongation of fibers. The as-spun mats were highly soluble in acidic and aqueous solutions. To produce insol- uble mat, post-production procedure featuring vapor-phase
glutaraldehyde (GA) can be taken to effectively cross-link thefiber mats utilizing Schiff base imine functionality.
This method of creating cross-linked chitosanfibers is a two-step method;fiber production followed by fiber exposure to vapor-phase GA for 24 h. The same authors[48]demonstrated that cross-linked chitosanfibers can be produced utilizing a one-step production meth- od instead of the previously reported two-step method. 50 wt.% GA in water solution is added to the chitosan/TFA system just prior to spin- ning. The cross-linking occurs during the time it takes to electrospin.
The implementation of a one-step as opposed to a two-step produc- tion method would lower manufacturing costs and production times for all applications where chitosanfiber mats would be used.
Hence, this reactive electrospinning method could bring chitosan nanofibrous mat technology closer to being implemented for large- scale production[48].
It was found that after cross-linking; the medium molecular weightfibers increased in diameter by an average of 161 nm, have a decreased Young's modulus of 150.8 ± 43.6 MPa, and were insoluble in basic, acidic, and aqueous solutions. Cross-linking resulted in in- creased brittleness, a color change, and the restriction offiber sliding that resulted in the loss of a pseudo-yield point.
Electrospun one-step crosslinked chitosanfibers has average di- ameter of 128 ± 40 nm. The averagefiber diameters for non-cross- linked and two-step cross-linked chitosanfibers were found to be 77 ± 29 nm and 172 ± 75 nm, respectively[16].
It is of interest to note also that the average one-step cross-linked fiber diameters are larger than non cross-linked fibers, but smaller than two-step cross-linked fibers. Therefore, the one-step cross- linking method produces faster cross-linked chitosan fibers with smaller averagefiber diameters relative to those produced when the vapor-GA two-step method was employed. As most bio- macromolecular nanofibers have been formed at the electric field range of 0.5–4 kV/cm, electric field higher than 3 kV/cm was required for the formation of a uniform chitosan nanofiber. At an electric field of 1 kV/cm spindle-like bead and thickfiber structure was formed.
When the electricfield was high enough to overcome the surface ten- sion of chitosan solution, particularly in the range of 3–4.5 kV/cm or more, uniform and homogeneous fibers were formed. However, above 4.5 kV/cm much thinnerfiber was produced with many bead defects possibly due to increased elongation force and instability of charged jet induced by the stronger electric field. Average fiber Fig. 2. SEM micrographs of chitin and deacetylated chitin (chitosan) nanofibrous matrix, before and after deacetylation reaction for 150 min at 100 °C[32].
M.Z. Elsabee et al. / Materials Science and Engineering C xxx (2012) xxx–xxx 5
diameters and size distribution decreased with increasing electric field and more bead defects appeared at 5 kV/cm or more[13].
Chitosan solution in 90% acetic acid solution with a molecular weight of 1.1 × 106g/mol results in a very high viscosity, which pre- vents the solution from elongating as a jet in the electricalfield of the electrospinning unit[49]. The low molecular weight chitosan so- lutions in aqueous 70–90% acetic acid produce nanofibers with appro- priate quality and processing stability. This suggests that low molecular weight chitosan align more effectively in the electromag- neticfield of the electrospinning unit. The authors attributed this to the fact that the average length of polymer chains is below the threshold required for entanglement coupling formation[49]. The av- erage diameter of this product is 140 nm and the standard deviation is 5. According to this study, the optimum conditions for producing these stable, high-quality chitosan nanofibers are: needle inner diam- eter of 0.7 mm, tip-collector gap 16 cm voltage 17 kV, and feed rate of 8 × 10− 2mg/h (1.6 mm3/h).
3.1. Electrospinning of chitosan derivatives
3.1.1. Hexanoyl chitosan
Chitosan can be chemically modified into soluble derivatives and thus increase its potential applications. Acylated derivatives of chito- san are soluble in chloroform, benzene, and tetrahydrofuran THF.
Among such derivatives hexanoyl chitosan (H-chitosan) was found to be anti-thrombogenic and resistant to hydrolysis by lysozome [50,51]. As a result, H-chitosan is a very interesting derivative of chi- tosan for use in biomedical applications. Successful preparation of H- chitosanfibers were reported by Neamnark et al.[52].
Electrospinning of H-chitosan in chloroform was conducted with concentrations ranging from 4 to 14%[17,18]. At low concentration (4%) only beads were obtained. By increasing the concentration, beads and fibers were obtained, while only at 14% concentration ribbon-likefibers were formed. The ribbon-like structure is probably a result of the rapid evaporation of chloroform during thefibers flight to the collecting plate. The averagefiber diameter (width) increased from about 0.64μm at the concentration of 6% w/v to about 3.93 μm at the concentration of 14% w/v. The increase in thefiber diameter with increasing H-chitosan concentration should be a result of the in- creased viscoelastic force that works against the Coulombic repulsion force and the decreased path length of the charged jet that reduces the on-flight time during which a charged jet segment is thinned down by the Coulombic repulsion force. The effect of applied voltage as well as the conductivity on thefiber diameters were also investi- gated and were found to increase with increasing both the voltage and the conductivity of the solution. Some pyridinum formate PF (ranging between 2.5 and 10%) was added to increase the conductiv- ity of the spinning solution. The addition of PF has a marked effect on the morphology of the obtainedfibers as shown inFig. 3. The SEM im- ages revealed that the surface of as-spun PF-free H-chitosanfibers was rough with minute pores being present on it (Fig. 3a). The surface of as-spun PF-added H-chitosanfibers was also rough, but without the presence of minute pores (Fig. 3b).
3.1.2. PEGylation of chitosan
Poly (ethylene glycol) (PEG) is an ideal graft forming polymer be- cause it is soluble in water and organic solvents and it has low toxic- ity, good biocompatibility, and biodegradability[53]; it is approved to be used in food, cosmetics, personal care products and pharmaceuti- cal[54]. Indeed, PEGylation of chitosan has been shown to improve the affinity to water or organic solvents. The reaction was carried out most commonly by reductive amination of chitosan with PEG- aldehyde in aqueous organic acid[55–58]. Water solubility was easily achieved at a degree of substitution (DS) as low as 0.2 for either de- rivative whereas, the PEG-N,O-chitosan at DS = 1.5 was soluble in or- ganic solvents, including CHCl3, DMF, DMSO and THF. Electrospinning
of all aqueous solutions of PEGylated chitosan produced only beads.
Sprayed droplets were observed with aqueous solutions of all PEG- N-chitosan via reductive amination. The main challenge offiber for- mation from PEGylated chitosan is most likely due to the limitation in inter-molecular chain entanglement. The high surface tension of water requires high voltage, therefore Triton X-100™, a nonionic surfactant to reduce the solution surface tensions, was added to the spinning aqueous solution to reduce its surface tension. However even with lower surface tension, the electrospinning still produced beads. Therefore, organic solvents and co-solvents were tried.
Electrospinning of 15% PEG-N,O chitosan from 75/25(v/v) THF/DMF co-solvents with 0.5% Triton X-100TM produced uniform nanofibers with diameters ranging from 40 nm to 360 nm and an average diam- eter of 162 nm. Electrospinning of PEG-N,O-chitosan at 25% in DMF producedfibrous structure intermixed with beads. Ultra-fine fibers with diameters ranging from 40 nm to 360 nm and an average diam- eter of 162 nm were efficiently generated from electrospinning of 15%
PEG-N,O-chitosan in 75/25 (v/v) THF/DMF cosolvents with 0.5% Tri- ton X-100™[58]. None of the aqueous solutions of PEG-N chitosan or PEG-N,O-chitosan alone could be electrospun intofibers.
3.1.3. Carboxymethyl chitosan (CMC)
Electrospinning of aqueous solutions (6–20% concentrations) of CMC at varying main chain lengths (Mv = 40, 89, and 405 kDa) and with DS in the range of 0.25–1.19 produced only droplets, even with added 0.5% Triton X-100 to reduce the surface tension. Failure of CMC to formfibers is believed to be due to inefficient chain entan- glement due to the rigid and extendedα-1,4 polyglycosidic chains.
Electrospinning of mixtures of CMC with several water-soluble poly- mers polyethylene oxide PEO (100 kDa), polyacrylamide, PAAM (5000 kDa), and polyacrylic acid, PAA (450 kDa) produced fibers with considerable amounts of beads entangled with them[59].
Mixing 20–80% poly (vinyl alcohol), PVA (124–186 kDa), 87–89%
hydrolyzed with CMC at 8.5 wt.% total concentration produced con- tinuous and uniformfibers (Fig. 4). The averagefiber diameters de- creased slightly from 210 to 170 nm on increasing the CMC content from 20% to 50% (Fig. 4a–c). Further increasing the CMC content to 70% and 80% reduced the electrospinning efficiency and led to larger and highly variedfiber sizes (Fig. 4(d) and (e)). These observations showed that, among the water-soluble polymer studied, PVA enabled the most efficient generation of CMC binary nanofibers with an equal mass of CMC at DS = 1.14.
Cross-linking by heat-induced esterification (at 140 °C for 30 min) rendered the CMC/PVAfibrous membranes insoluble in water. The mass retention andfiber morphology confirmed that the more highly substituted CMC achieved more favorable inter-molecular crosslinking that led to a more stable and water-insolublefibrous membrane.
Fig. 3. Surface morphology of the as-spunfibers 8% w/v hexanoyl chitosan solution in chloroform (a) without addition of PF salt and (b) with 7.5% w/v PF salt addition. The applied electricalfield strength was 12 kV/12 cm[52].
3.2. Electrospinning of chitosan blends
3.2.1. Chitosan-poly (vinyl alcohol) blend
Since PVA is known to be non-toxic, water-soluble, biocompatible, and biodegradable synthetic polymer, which is widely used in bio- medicalfield and has excellent fiber forming ability and highly hydro- philic properties, it is to be expected that a membrane composed of nanofibers of PAV/CS blend produced by electrospinning could have an important role in the biomedicalfield[60].
Ohkawa et al.[46]have mixed chitosan with poly (vinyl alcohol) (PVA), which can interfere with the rigid association of the chitosan molecules. PVA was chosen because it strongly interacts with chito- san through hydrogen bonding on a molecular level[61,62]and it can be conveniently electrospun from an aqueous medium[63,64].
Two chitosan samples were used (viscosity average molecular weight, Mv = 2.1 × 105and degree of deacetylation, 0.78) and the sec- ond was chitosan 100 (Mv = 1.3 × 106; degree of deacetylation, 0.77) with PVA (degree of polymerization, approximately 2000; number average Mn = 8.8 × 104). The electrospinning experiments were per- formed at room temperature. The electrospinning was conducted by dissolving the PVA in distilled water at a concentration of 9 wt.%, and chitosan was dissolved in neat formic acid (FA) at 7 wt.%.Fig. 5 shows SEM photographs of the chitosan/PVA blended electrospun fabrics. When a small portion of the PVA was mixed with chitosan (chitosan:PVA = 90:10), as presented inFig. 5a, beads were deposited on the collector. As the ratio of chitosan in the solution decreased (chitosan:PVA = 70:30), the size of the beads became smaller and thinfibers coexisted among the beads (Fig. 5b). When equal volumes of the chitosan and PVA (50:50) solutions were blended, homoge- nous fibers with an average diameter of 120 nm could be spun (Fig. 5c; diameter distribution, 83–170 nm). At a chitosan:PVA ratio of 30:70 thefibers were thicker (Fig. 5d, average diameter, 170 nm diameter distribution, 110–220 nm) than those prepared at 50:50 (Fig. 5f; average diameter, 170 nm; diameter distribution, 120–220 nm)[46].
In another study, high molecular weight PVA (Mw = 124–186 kDa, 87–89% hydrolyzed) was added to a chitosan in 2% v/v aqueous acetic
acid solution to moderate the repelling interaction between the poly- cationic chitosan molecules and to enhance the molecular entangle- ment[65]. Fiber formation could be sustained from electrospinning of mixtures with chitosan/PVA mass ratio ranging from 11/89 to 50/
50 at decreasing total polymer concentrations from 6% to 3%. Thefi- bers electrospun from the 6% mixtures of chitosan/PVA at 11/89 or 17/83 mass ratio were of varying sizes. Thefibers were as thin as 100 ± 20 nm in diameters and interspersed with enlarged spindle- like sections of about 500 ± 100 nm in widths. The average fiber length between the spindle-like structures was 10 ± 3μm. As the chi- tosan/PVA mass ratio increased to 25/75 (4% solution), the enlarged sections became more bead-like structures in larger numbers. Thefi- bers between the beads were muchfiner, about 20±5 nm in diameter and 7 ± 3μm in length, which was likely due to the lowered polymer concentration. The mixture with 50/50 chitosan/PVA generated signif- icantly fewerfibers, but some very larger micrometer size beads.
From this study, it can be concluded that the addition of PVA facil- itated the electrospinning of chitosan into nanofibers, but only with up to 25% chitosan. Also,fiber formation was improved with the chi- tosan hydrolyzed for just 2 h (1500 kDa) in comparison to the origi- nal chitosan (1600 kDa) at the same equal mass composition. The total polymer concentration suitable forfiber formation increased from 3% to 5% with longer hydrolysis time which reduced the mol wt from 1500 kDa to 800 kDa. With the latter chitosan,fiber forma- tion could be sustained continuously for several hours at a rate of 60 mg in dryfiber mass per hour. The average fiber diameter was around 50 ± 10 nm and with very few beads.
Electrospinning of poly (vinyl alcohol) (PVA) (Mn= 9.4 × 104, and degree of hydrolysis = 96%)/chitosan (CS), DDA = 78 nanofibrous membranes with different weight ratio of PVA to CS was conducted by Jia et al. [60]. Both PVA and CS were dissolved in distilled water forming 20% and 3% by wt, respectively, and then the solutions were mixed in different ratios and electrospun as such. The concentration of PVA/CS blend solutions was from 3 wt.% to 9 wt.%. It was found that the morphology and diameter of the fibers were also strongly influenced by the ratio of CS in the blend. The fiber diameter was found to decrease gradually Fig. 4. Fibers electrospun from 8.5 wt.% aqueous mixtures of CMC (Mv = 405 kDa, DS = 1.14)/PVA at mass ratios of: (a) 20/80, (b) 30/70, (c) 50/50, (d) 70/30 and (e) 80/20[59].
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with increasing CS content in the blend. However when CS percent was higher than 30% nofiber formation was observed which had been observed also by Ohkawa et al.[46]. The authors attributed this to the increased charge density on the surface of the ejected jet due to the cationic nature of CS. This indicated that the repul- sive force between ionic groups within the polymer backbone was expected to inhibit the formation of continuous fiber during electrospinning. Similarfindings were reported also by Zong et al.
[66] and Park et al. [67]. Regarding the polymer concentration, the average diameter of the fibers increased with increasing the concentration. Beads were only generated by electrospinning the CS/PVA blend solution at the concentration below 3 wt.%. In con- trast, when the concentration of PVA/CS blend solution was more than 10 wt.%, the electrospinning process was hard to maintain due to the high viscosity of the solution. Therefore the optimum concentration of the electrospinning blend should be in the range 7–9 wt.%.
3.2.2. Crosslinked chitosan/PVA blend
A photoinitiator has been used to crosslink PVA/CS nanofibers after irradiation with UV light [68]. Polyethyleneglycol-600- dimethylacrylate (PEGDMA) and photoinitiator 2-hydroxy-1-[4-(2- hydroxyethoxy) phenyl]-2-methyl-1-propanone (HEPK) were added into the spinning solution of CS/PVA blend and then the obtainedfi- bers were subsequently irradiated with the UV rays.Fig. 6 shows the SEM images of the nanofibers noncrosslinked (a) and crosslinked by PEGDMA/HEPK with different contents (b–d). After UV irradiation, the morphology of electrospunfibers did not change in comparison with noncrosslinkedfibers [seeFig. 6(e–g)]. The fibers were cylindri- cal and smooth with average diameters ranging from 200 to 800 nm.
These results implied that the photocrosslinked electrospunfiber membranes could provide a highly porous structure for cell attach- ment and proliferation in the wet stage[50,68]. It is also implied that multi-scale bi-modelfibers could be electrospun successfully in a single step, producing structures that have potential applications in differentfields.
Submicronfibers of poly vinyl alcohol (PVA), chitosan oligosac- charide (COS), and montmorillonite clay (MMT) composite were electrospun from aqueous solutions[69].
The PVA/COS ratio and MMT concentration were found to play im- portant roles in nanofiber mat properties. XRD and TEM data demon- strated that exfoliated MMT layers were well-distributed within the nanofiber. It was also found that the mechanical property and ther- mal stability were increased with increasing the COS and MMT contents.
3.2.3. Chitosan–polyethylene oxide blends
Polyethylene oxide was chosen as a co-spinning agent due to its excellent electrospinning characteristics, its ability to form ultrafine fi- bers, its linear structure withflexible chains, its biocompatibility, its solubility in aqueous media, and its capability to form hydrogen bonds with other macromolecules. Three molecular weights of chito- san (600,000, 400,000 and 148,000 g/mol) blended with polyethylene oxide PEO were fabricated by electrospinning to defect free nanofibers with average diameters ranging from 62 ± 9 nm to 129 ± 16 nm[70].
The chitosan samples had a degree of deacetylation of 75–85% and were made in concentrations from 1 to 8 wt.% in increments of 1 wt.%.
The replacement of some of the chitosan with PEO while maintaining the same total polymer concentration allows the solution as a whole to be more easily electrospun by offsetting chitosan's high viscosity in the acetic acid/water solvent system[71]. When the total polymer concentration was increased from about 2.5 to 3.4 wt.%, while keeping the ratio of chitosan to PEO constant at (2:3), nearly defect free nanofibers were obtained. In order to further increase the amount of chitosan in the nanofibers, the concentration of chito- san in the blended solutions was increased from 1.6 to 2.5 wt.%. This change increased the total amount of polymer in the solution from 3.4 to 4.0 wt.%. Defect-free nanofibers of over 60% chitosan were pro- duced. It has been found also that reducing the acetic acid concentra- tion from 45% to about 30% reduced the number of bead defects in the electrospunfibers, possibly by altering the conformations of the poly- mers and the conductivity of the solutions.
Fig. 5. SEM photographs of the chitosan and PVA blended electrospunfibers. The volume ratios were chitosan10:PVA=90:10 (panel a), 70:30 (panel b), 50:50 (panel c), 30:70 (panel d) and 0:100 (panel e). Chitosan100 was dissolved in formic acid (or 0.2 M acetic acid) at 2 wt.% and the solution was mixed with 9 wt.% PVA in a volume ratio of 50:50, then the mixed solution was electrospun (panel f)[46].
A recent work describes the effect of two parameters on the electrospinning behavior of chitosan/polyethylene oxide blends [72]. The effect of molecular weight and acidity in the presence of a nonionic polysorbate surfactant (Tween 20) to improve functionality of the resulting nanofibers has been investigated. The presence of sur- factant resulted in decrease of surface tension and in the formation of smooth or beadedfibers. However, the main conclusion of this work was that acetic acid concentration strongly influenced the chain en- tanglement of chitosan and consequently the properties of chitosan solutions and the morphology of the formed fibers; a conclusion that has been given by many other authors.
Nanofibers were fabricated by electrospinning a mixture of cation- ic chitosan and neutral polyethylene oxide (PEO) at a ratio of 3:1 in aqueous acetic acid[73]. In this blend, PEO acts as a plasticizer facili- tating orientation andflow of chitosan by uncoiling and wrapping around chitosan chains[74–76].
In order to further modify the quality and morphology of the nanofibers, Zeng et al. [77] have added several surfactants to the electrospinning solutions. The addition of ionic surfactants to pol- yionic polymers as chitosan alters its solution properties such as vis- cosity, conductivity and surface tension. In turn, these changes in solution properties alter the Taylor cone formation, jet expulsion and jet bending/whipping, influencing the type, structure and dimen- sions of the nanostructures formed. Different types of surfactants are used in this study; anionic sodium dodecyl sulfate (SDS), nonionic polyoxyethylene glycol (23) lauryl ether (Brij 35) and cationic dode- cyl trimethyl ammonium bromide (DTAB). Surfactants and polymers above their critical micelle concentrations were dissolved in aqueous acetic acid followed by addition and dissolution of chitosan and PEO.
The observed rheological behavior of polymer solutions in the presence of surfactants suggests that polymer–polymer interactions are modulated by the presence of surfactants and the type of solvent in which the polymers are dissolved. Chitosan in acetic acid carries a strongly positive charge it can thus be expected to interact electro- statically with ionic surfactants such as SDS and DTAB. Since solutions were added above their critical micelle concentrations, micelles may interact with the polymer chains.
SDS micelles, which are negatively charged, could bind to one or more individual chitosan molecules. This couldfirstly decrease the re- pulsive interactions between individual chitosan chains facilitating increased entanglement, and secondly lead to bridging between poly- mer chains which may explain the increasing viscosity upon addition of SDS to chitosan and chitosan–PEO blend solutions. It will also lead to decreasing the surface tension of the solution favoring electrospinning.
DTAB on the other hand is a positively charged surfactant whose head groups should be electrostatically repelled from the cationic groups on the chitosan backbone. Nevertheless, DTAB may still bind to polymers through hydrophobic interactions between its non- polar tail and any non-polar groups on the polymer chain. If binding occurred, there would be an increased electrostatic repulsion be- tween chitosan/DTAB complexes thus decreasing entanglement and viscosity.
Brij 35 as a nonionic surfactant would be expected to show no electrostatic interaction and instead may interact with polymers sole- ly via hydrophobic binding. The fact that addition of Brij 35 to the polymer solutions caused little change in viscosity suggests that it did not strongly affect the interactions between the polymer mole- cules[77].
When surfactants of any type were added to the chitosan solution, the presence of bead defects was not completely prevented but the onset of nanofiber fabrication was greatly improved. Using Brij 35 resulted in the formation of a small amount of ultrafine fibers sur- rounded by many beads. In the case of SDS, needle-likefibers possibly composed of SDS crystals and chitosan were deposited on the collec- tor plate, while DTAB yielded structures with a beaded-string appearance.
With addition of PEO to chitosan, a well defined Taylor cone was formed, a jet was obtained and a deposition of an interesting nanofibrous structures with nanofibers having average diameters ranging from 10 to 250 nm was obtained. When Brij 35 was added to the polymer blend solutions in 50% acetic acid, smooth nanofibers with diameters varying from 70 to 120 nm were collected. Some long and branchedfibers were observed which is indicative of the split of a Fig. 6. SEM images of electrospunfibers of CS/PVA with different content of PEGDMA/HEPK: (i) after UV irradiation (a) 0 wt.%, (b) 20 wt.%, (c) 35 wt.%, (d) 50 wt.%; (ii) before UV irradiation (e) 20 wt.%, (f) 35 wt.%, (g) 50 wt.%[68].
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singlefiber during the electrospinning process. Addition of SDS to the polymer blends yielded nanofibers of relatively small diameters (10–60 nm), which was the smallest size of nanofibers collected in the experiment, albeitfibers had some minor bead defects. Fiber di- ameter ranged from 50 to 130 nm in composite samples that con- tained the positively charged DTAB. Similar nanofiber structures were obtained using 90% acetic acid solution.
The authors[77]concluded that, addition of surfactant can help induce formation of a polymer jet but may simultaneously alter the relative concentration of polymers in the generated nanofiber.
Hence, the concentration of one of the polymers in thefibers may be substantially lower than in the original polymer solution. The fact that surfactant was co-spun with the blend could make it possible for micelles to remain intact and become part of the nanofibers formed. Since micelles can be loaded with lipophilic functional ingre- dients, this could serve as an additional means to further fun- ctionalizefibers and broaden the number of applications in which nanofibers could be used[77].
Addition of metal ions to the chitosan/PEO blends was found to decrease the viscosity (45–60%) and hydrogen bonds of the blends [78]. Scanning electron micrographs confirmed that the presence of 0.4–1.6 wt.% NaCl (or KCl) in the blend solutions produced by electrospinning nanofibers, accompanying re-crystallization of inor- ganic salts, while addition of appropriate amounts of CaCl2or FeCl3
(0.8 wt.%) resulted in beneficial effect on defect-free nanofibers. The authors concluded that this general electrospinning strategy, which involves doping of metal ions, may be versatile to modulate thefi- brous structure and functionality of polymers.
3.2.4. Collagen–chitosan blend
Collagen is a polyprotein that has been widely used for tissue- engineering scaffolding, owing to a wealth of merits such as biological origin, non-immunogenicity, excellent biocompatibility and biode- gradability[79]. Chitosan can form a complex with collagen[80,81]
and can modify the properties of collagen when the biological or me- chanical properties are considered. The electrospun chitosan–colla- gen complex may represent a potential ideal tissue engineering scaffold and a promising functional biomaterial.
The collagen–chitosan complex can be used to mimic the compo- nents of the native extracellular matrix. It has also been reported that the hybrids of collagen–chitosan manufactured by crosslinking, wet/
dry spinning and freeze-drying have biological and mechanical bene- fits to be used as the tissue engineering scaffold[82,83]. The influence of chitosan on the physicochemical and biochemical properties of col- lagen has been studied by many authors[84–87]. It seems that a new hydrogen bonding network appear in the blend that alter the collagen helical character and therefore the overall physical parameters of the blend. The molecular interaction in the collagen–chitosan complex was characterized by FT-IR spectroscopy and an indication that a new complex has been formed between the collagen and chitosan has been observed. The electrospinning of chitosan–collagen complex has been studied by Zonggang Chen et al.[88]. Three kinds of sol- vents, 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP), trifluoroacetic acid (TFA) and dichloromethane (DCM) were used to adjust the best con- dition for electrospinning. The solvent choice was based on a previous experience where chitosan was electrospun using the TFA/DCM sol- vent mixture, while TFA was used for collagen electrospinning. Colla- gen–chitosan complexes with different weight ratios (w/w: 80:20, 50:50 and 20:80) were electrospun in the selected solvent of HFIP/
TFA (90/10) at a concentration of 10%.
The mean diameters of thefibers decreased with increasing the chitosan content in the collagen–chitosan complex. The reason may be that the organic salts formed between TFA acid and the amino groups on chitosan had increased the charge density of the polymer solution. It has been reported that addition of small amounts of or- ganic salt to the spinning solution may reduce thefiber diameter[77].
3.2.5. Chitosan–agarose blend[89]
Agarose, a neutral polysaccharide obtained from red algae, has been extensively used in food, cosmetics and pharmaceutical indus- tries[90]. Agarose is a linear polymer, made up of the repeating mo- nomeric unit of agarobiose which is a disaccharide made up ofD- galactose and 3,6-anhydro-L-galactopyranose (seeScheme 1).
The trifluoroacetic acid (TFA)/dichloromethane (DCM) mixture (7/3, v/v) was used as a suitable solvent for both chitosan and aga- rose, with a 7% (w/v) chitosan–agarose complex concentration. The weight ratios of chitosan to agarose were chosen as 70:30, 50:50, and 30:70. It was found that the addition of agarose dramatically re- duces the viscosity of the blend. Compositional analysis by FT-IR rev- ealed the presence of the strong interaction and good compatibility between the chitosan and agarose components[89].
Morphological observation of the electrospun chitosan/agarosefi- bers revealed that pure chitosan solution in TFA/DCM produced smooth, continuous, and randomly orientedfibers, the results were in good agreement with Matsuda et al. [91]. The fine, cylindrical nanofibers were also successfully generated from the chitosan/aga- rose blend with 30% and 50% agarose, respectively. Moreover, the hy- bridfiber displayed a smooth and uniform surface, indicating that chitosan and agarose were homogeneously mixed. However, when 70% agarose was contained in the blend, beads andfibers coexisted in the SEM image. It was also observed that the addition of agarose led to a significant decrease in the fiber diameter. The pure chitosan fibers exhibited a wide diameter distribution ranging mostly from several hundreds of nanometers to 2.5μm. Their average fiber diam- eter was calculated to be approximately 1.76 ± 0.59μm. With the ad- dition of agarose, however, the average fiber diameters were substantially decreased to about 520 ± 35 nm and 140 ± 9 nm at 30% and 50% agarose, respectively, and thereafter sustained at 70%
agarose.
3.2.6. Chitosan/polyacrylamide
Polyacrylamide, PAAm is a hydrophilic, high molecular weight synthetic polymer which like chitosan has\NH2groups on its side chain and can form hydrogen bonds with other polymers. PAAm has been widely used for waste water treatment as aflocculent to bind heavy metal ions by forming coordination bonds and cationic poly- acrylamide has also been used for anti-microbial applications[92].
PAAm (MW = 5000 kDa) has been blended with HMW chitosan (Mv = 1400 kDa with 80% DDA) and a nonwoven fibers without bead defects were obtained by electrospinning using a modified electrospinning unit wherein polymer solutions can be spun at tem- perature greater than ambient to 100 °C[93].
This study shows that electrospun blend solutions containing 95%
chitosan leads to poorfiber formation at room temperature and very fewfibers are collected on the target. By increasing the temperature, fiber formation is improved with bead-less fibers formed at 70 °C.
When chitosan content was reduced to 90% with increasing spinning temperature, a transformation from beadedfibers to uniform bead- fewer fibers took place. Further reduction to 75% chitosan in the blend leads to formation of bead-lessfibers at room temperature. As spinning temperature is increased, an increase infiber diameter is ob- served[92]. At higher temperatures, there is a faster evaporation of solvent leading to faster drying of the charged jet and increased
Scheme 1. The structure of an agarose polymer[90].
chain entanglements. These nanofibers showed high binding efficien- cies for Cr IV (4 mg chromium/g chitosan).
3.2.7. Chitosan/zein blend
Zein, a major protein of corn, has been used in the food industry as a coating material[94]. Zein possesses the additional benefits of being renewable and biodegradable. Zein microspheres have also been inves- tigated for use as carriers to protect drugs from stomach acid. Dong et al.
[95]reported that zein was a promising biomaterial with good biocom- patibility. Itsfibers have also been produced by electrospinning from acetic acid, aqueous methanol, ethanol and isopropyl alcohol. Alco- hol solutions producedfibers that were predominantly ribbons. Fi- bers spun from acetic acid solution have a round morphology with a narrower distribution of diameters when spun under suitable conditions[96].
Zein/chitosan blends with different compositions 99:1, 97:3, 95:5 and 90:10 have been prepared with a total polymer concentration of 25 wt.% and were electrospun from ethanol:trifluro acetic acid solution (2:1 wt/wt) a flow rate of 0.20 ml/h 14 kV of voltage and tip-to- collector distance of 10 cm was set as the electrospinning conditions.
FTIR studies indicated some interaction between zein and chitosan which may suggest that dissolving in a common solvent followed by electrospinning could enhance molecular interactions, in spite of previous reports of incompatibility of similar systems[97]. The blends containing below 5 wt.% of chitosan in the formulation generated clear continuous ultrathin fiber morphologies. Pure zein ultrathin fibers, originated from electrospinning of TFA solutions, presented similar ribbon-likefiber morphologies (with average cross-sections of 320.9 ± 92.3 nm) as those reported earlier for the same system electrospun from alcohol solutions[98]. When 1 wt.% chitosan, was blended with zein, similar ribbon-likefiber morphologies, but with smaller average diameter (192.3 ± 54.4 nm), were deposited on the col- lector. As the ratio of chitosan increased to 3 and 5 wt.%, the shape of the fibers became even smaller (with main cross-sections of 161.7±39.6 and 128.5±26.2 nm, respectively) but also exhibited increasing pres- ence of beaded regions (with mean diameters of 315.6 ±76.4 and 420.4±86.7 nm, respectively). When the chitosan content reached 10 wt.%, the beaded morphology became dominant (with average mean diameter of 598.2±153.5 nm) but these beaded structures were seen to be interconnected by very ultrathin nanofibers (with mean cross-section of 36.1±12.1 nm). Thefiber mats presented a smooth sur- face and higher magnification of the material did not provide evidence of a phase separated morphology[97].
A selective dissolution of the chitosan fraction was carried out by washing thefiber mats with dilute aqueous acetic acid (1 wt.%), a solvent which earlier tests proved to selectively dissolve only chito- san. After washing thefibers, the solvent was allowed to evaporate at room temperature and the samples were immediately observed by SEM (seeFig. 7). This image indicates that a homogenization of
thefibrillar morphology and a reduction of the beaded regions is ob- served, suggesting that, on one hand, thefibers are water resistant which give them added bonus on moisture contact applications and justifies the selection of zein as matrix, and on the other hand, implies that chitosan seems to be preferentially placed in the beaded regions and hence causes bead formation due to its particular characteristics such as higher surface tension. The washing acid treatment was also found to lead to somewhat thicker or swollen structures, due to prob- ably moisture soaking and solvent-inducedfiber coalescence.
This study pointed out that novel antimicrobial material based on ultra thin structured zein and in blends of zein with chitosan, has been developed and found to have potential applications in different fields. The biocide properties of the electrospun ultra thin structured systems were assessed and it was found that even a small amount of chitosan (1 wt.%) can induce strong antimicrobial effect against Saurueus which was related to the presence of chitosan. Electrospun fibers of these biomaterials could therefore be of great interest in var- ious applications such as active food packaging and in pharmaceutical and biomedical applications, for instance tissue engineering, medical implants and body-implant interphases[97].
3.2.8. Nylon-6/chitosan blends
Composite membranes of nylon-6/chitosan nanofibers with differ- ent weight ratio of nylon-6 to chitosan were fabricated successfully using the electrospinning technique[99]. The study concluded that morphology and diameter of the nanofibers were influenced by the concentration of the solution and weight ratio of the blending compo- nent materials. Furthermore FT-IR analysis demonstrated an IR band frequency shift that appeared to be dependent on the amount of chi- tosan in the complex. Observations from XRD and DSC suggested that a new fraction ofγ phase crystals appeared and increased with the in- creasing content of chitosan in blends, this indicated that inter- molecular interactions occurred between nylon-6 and chitosan.
Results from performance data in mechanical testing showed that intermolecular interactions varied with varying chitosan content in thefibers. Thus, a new composite product was created and the stabil- ity of this system was attributed to strong new interactions such as hydrogen bond formation between the nylon-6 polymers and chito- san structures.
4. Quaternized chitosan blends 4.1. Quaternized chitosan/polylactides
Quaternized chitosan (QCh) derivatives have shown higher activ- ity against bacteria, broader spectrum of activity and higher killing rate as compared to those of chitosan[100,101]and thus are potential candidates for wound dressing applications.
A one-step method—electrospinning in common solvent has been used for the preparation of novel bicomponent hybrid nanofibrous materials based on natural chitosan or its quaternized derivative (N, N,N trimethyl chitosan iodide, shown inScheme 2) and synthetic al- iphatic polyester Poly[(L-lactide)-co-(D,L-lactide)] PLA[100].
The spinning solution CS/PLA was prepared by mixing 5 wt.% chi- tosan solution in TFA/DCM (70/30 vol.%) with 5 wt.% PLA solution in TFA/DCM (70/30 vol.%) at weight ratio of CS:PLA = 50:50. The spin- ning solution QCh/PLA was prepared by mixing 5 wt.% QCh solution
Fig. 7. Typical SEM photographs of electrospunfiber mats obtained from ethanol–TFA solutions of (a) 95/5 wt.% zein–chitosan and (b) 95/5 wt.% zein–chitosan blend washed
with aqueous acetic acid solution (1 wt.%)[97]. Scheme 2. Quaternized chitosan (QCh)[101].
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in dry DMF/DMSO (60/40 vol.%) with 5 wt.% PLA solution in dry DMF/
DMSO (60/40 vol.%) at weight ratio of QCh:PLA = 30:70.
In order to stabilize the electrospun bicomponent CS/PLA and QCh/PLA mats against dissolving in water, the nanofibers containing chitosan or quaternized chitosan were cross-linked with glutaralde- hyde vapor in order to enlarge the possibilities for their biomedical applications. The crosslinking of chitosan nanofibers with glutaralde- hyde vapor proceeds with Schiff base formation and without forma- tion of Michael-type adducts with terminal aldehyde group. In order to neutralize the protonated amino groups of chitosan component in CS/PLA mats by TFA, they were exposed to ammonia vapor for 40 min.
As it is seen fromFig. 8, the effect on the morphology of the crosslinked CS/PLA nanofibers (CS:PLA=50:50 wt.%) after soaking in acidic aqueous solution (0.2 M CH3COOH), and the hybrid crosslinked nanofibrous mats maintained fiber morphology and retained the integrity of thefibrous structure, although they were swollen.
The antibacterial activity of crosslinked bicomponent CS/PLA and QCh/PLA nanofibers against Gram positive bacteria S. aureus and Gram negative bacteria E. coli was evaluated by counting the viable bacteria cells that rested in a bacteria suspension after the contact of electrospun mats with the suspension. These hybrid nanofibers showed the capability to kill all the S. aureus and E. coli cells within 60 min of contact[101].
4.2. Quaternized chitosan/poly (vinyl alcohol)
Nano-fibers containing quaternized chitosan (QCh) have been prepared by electrospinning of QCh solutions mixed with poly (vinyl alcohol) (PVA) [102]. The quaternized Chitosan used was N-Butyl-N, N-dimethyl chitosan iodide prepared by Kim et al. meth- od[103]. The spinning solutions QCh/PVA were prepared by mixing 8% aqueous PVA solution with 8% aqueous solutions of QCh at weight ratios of QCh:PVA = 1:4, 2:3, 1:1, 3:2 and 4:1. The total polymer concentration in the mixed solutions was 8 wt.%. The solu- tions for the preparation of cross-linked nanofibers and of solution- castfilms were prepared using triethylene glycol diacrylate TEGDA (10.7%) a crosslinking agent, 2,2-dimethoxy-2-phenylacetophenone DMPA (1%) in DMSO and ammonium peroxydisulfate (1%) acting as photoinitiator (all in weight percent to total polymer content)
were added to the solution QCh/PVA at weight ratio of QCh:
PVA = 2:3. The total polymer concentration in the mixed solutions was 10 wt.% (H2O/DMSO = 92:8 w/w). The crosslinking of the electrospun mats or thefilms was performed by irradiation with a UV lamp (365 nm, 200 W).
It was found that crosslinking of the nanofibers increases their water resistance and decreased the fiber diameters from 217 to 116 nm. The authors explained the observed effects as due to the increase in the conductivity of the solution from 2.95 to 4.3 mS/
cm when the inorganic salt-ammonium peroxide is added. It has been reported that the increase of the charge density and of the conductivity of the solution resulting from the presence of a salt or low-molecular-weight compound with ionizable groups leads to the decrease of the diameter of the electrospunfibers and to a smaller number of defects [104–106]. Stronger elongation forces are imposed to the jet due to the repulsion of the excess charges, thus resulting in the formation of nano-fibers with smaller diameters.
Microbiological screening has demonstrated the antibacterial ac- tivity of the QCh/PVA as well as the photo-cross-linked electrospun nanofibers against S. aureus and E. coli. The test showed that MIC of QCh against E. coli was 2070μg/ml. The electrospun QCh/PVA mats were exposed also to Gram-positive bacteria E. coli. The reduction of bacteria E. coli for photo-crosslinked QCh/PVA nanofibers con- taining 2885μg QCh was 98% after 120 min contact time. The obtained nano-fibrous electrospun mats are promising for wound- healing applications because it could contribute to the prevention of secondary infections in wounds by S. aureus, resulting in limited scar formation.
Another chitosan quaternary salt N-[(2-hydroxy-3- trimethylammonium) propyl] chitosan chloride (HTCC), a water- soluble derivative of chitosan, was synthesized via the reaction be- tween glycidyl–trimethyl ammonium chloride and chitosan [107].
Solutions of PVA–HTCC blends were electrospun. The average fiber diameter was in the range of 200–600 nm. SEM images revealed that increasing HTCC content in the blends decreases the average fiber diameter. Shear viscosity of the PVA–HTCC blend decreases as HTCC content increases in the blend, whereas the electrical con- ductivity of the blend increases. Electrospinning voltage had a sig- nificant effect on the reduction of the electrospun fiber diameters.
Electrospun nanofibrous PVA–HTCC mats showed a good antibacterial activity against the Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus. This feature suggests that the PVA–HTCC electrospun mats are potentially good candidates for biomedical applications.
4.3. Quaternized chitosan/poly(vinyl pyrrolidone)
The quaternized chitosan QCh in Scheme 2 has been also electrospun in a blend with poly (vinyl pyrrolidone) PVP[108]. The latter polymer has many useful properties including non-toxicity, biocompatibility, high hydrophilicity, good complexation properties, and film-forming ability [109]. PVP has also been successfully electrospun into nanofibers from ethanol, DMF, dichloromethane or their mixtures [110]. The QCh/PVP system is very similar to the QCh/PVA system investigated by the same authors. The spinning solu- tions QCh/PVP were prepared by mixing 20 wt.% aqueous PVP solu- tion with 20 wt.% aqueous solutions of QCh at weight ratios of QCh:
PVP = 1:4, 2:3, 1:1, 3:2 and 4:1. The total polymer concentration in the mixed solutions was 20 wt.%. Cylindrical continuous defect-free fibers were obtained with the whole studied ratios with an average diameter in the range from 1530 to 2800 nm. An increase in the aver- age diameters was observed on increasing the appliedfield strength (AFS) from 1.6 to 2.8 kV/cm keeping a constant ratio of the polymer blends, contrary to the previously studied (QCh/PVA) system[111], Fig. 8. Nano-fibers mat (CS:PLA=50:50 wt.%) treated with ammonia vapor for 40 min
and crosslinked with GA vapor for 4 h (a), and after contact with 0.2 M CH3COOH for 10 h (b)[101].
