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*Author for c

doi:10.1098/rsif.2012.0241Published online

Received 23 MAccepted 30 A

New crosslinkers for electrospunchitosan fibre mats. I.

Chemical analysisMarjorie S. Austero1, Amalie E. Donius1, Ulrike G. K. Wegst1,2

and Caroline L. Schauer1,*1Department of Materials Science and Engineering, Drexel University,

3141 Chestnut Street, Philadelphia, PA 19104 USA2Thayer School of Engineering, Dartmouth College, 14 Engineering Drive,

Hanover, NH 03755 USA

Chitosan (CS), the deacetylated form of chitin, the second most abundant, natural polysac-charide, is attractive for applications in the biomedical field because of its biocompatibilityand resorption rates, which are higher than chitin. Crosslinking improves chemical and mech-anical stability of CS. Here, we report the successful utilization of a new set of crosslinkers forelectrospun CS. Genipin, hexamethylene-1,6-diaminocarboxysulphonate (HDACS) and epi-chlorohydrin (ECH) have not been previously explored for crosslinking of electrospun CS.In this first part of a two-part publication, we report the morphology, determined by fieldemission scanning electron microscopy (FESEM), and chemical interactions, determinedby Fourier transform infrared microscopy, respectively. FESEM revealed that CS could suc-cessfully be electrospun from trifluoroacetic acid with genipin, HDACS and ECH added to thesolution. Diameters were 267+ 199 nm, 644+ 359 nm and 896+ 435 nm for CS–genipin,CS–HDACS and CS–ECH, respectively. Short- (15 min) and long-term (72 h) dissolutiontests (T600) were performed in acidic, neutral and basic pHs (3, 7 and 12). Post-spinning acti-vation by heat and base to enhance crosslinking of CS–HDACS and CS–ECH decreased thefibre diameters and improved the stability. In the second part of this publication, we reportthe mechanical properties of the fibres.

Keywords: electrospinning; chitosan; biopolymer; genipin; diisocyanate;epichlorohydrin

1. INTRODUCTION

Biopolymers, such as chitin and chitosan, are excellentcandidates for a wide variety of applications, especiallyin the biomedical field, because they are renewable[1–3], biodegradable and biocompatible [4,5]. By formingfibres and fibre mats, the material’s surface-to-volumeratio can be markedly increased. The resulting increasein chemical functionality makes biopolymer fibres andmats highly attractive for food-processing and biomedicalapplications, specifically in the areas of active food packa-ging and filtration, tissue engineering and wound healing[6]. Electrospinning is a well-established technique withwhich fibres and mats can be manufactured in a simpleand flexible process. Polymer solutions are placed in asyringe which is connected to a conductive collectorand a high voltage source. Fibres and mats are spunfrom the solutions by applying the appropriate accelerat-ing voltage to overcome the solution’s surface tension.Their properties such as fibre diameter, mat porosityand morphology can be carefully tailored for a givenapplication through the electrospinning parameters

orrespondence (cschauer@coe.drexel.edu).

arch 2012pril 2012 1

such as the applied electric field strength, solution flowrate and tip-to-collector distance [7–9].

Chitin (N-acetyl-D-glucosamine), the second mostabundant, naturally occurring polysaccharide, is thestructural polymer of exoskeletons of crustaceans (suchas crabs and shrimps) as well as squid pens [3,5,10,11].When compared with chitin, chitosan (CS), the deacety-lated form of chitin, is widely used in the food-processingand biomedical fields owing to its increased solubility inaqueous acid solutions.

CS has successfully been electrospun with a number ofcopolymers such as polyethylene oxide (PEO) [12] andpolyvinyl alcohol (PVA) [13] using a variety of solventsystems; it has also been spun solo [14–18]. NeutralizedCS fibre mats are non-toxic and biocompatible, andtherefore have great potential for its use as filtrationmembranes and tissue engineering scaffolds. Because inthese applications, membranes and scaffolds not onlyhave to be stable in a wet environment, but also resistantto a combination of chemical and mechanical stresses,they need to be stabilized.

Crosslinkers are agents that stabilize polymersthrough the coupling and bonding of functional groupsin the chains, thereby preventing dissolution. In the

This journal is q 2012 The Royal Society

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Figure 1. A schematic of the select functional groups of: (a) chitosan, reacting with: (b) genipin, (c) HDACS, (d) ECH and (e) GAresulting in the formation of crosslink products: ( f ) CS–genipin, (g) CS–HDACS, (h1,h2) CS–ECH and (i) CS–GA,respectively.

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form of beads, films and membranes, the crosslinking ofCS has been reported with crosslinkers such as glutaral-dehyde (GA) [18–21], genipin [22–24], diisocyanates[25,26] and epoxides [27]. Recently, Schiffman andSchauer [19] reported the successful one-step crosslinkingwith GA of CS fibres electrospun from trifluoroaceticacid (TFA), and their improved chemical stability andmechanical properties.

GA (figure 1e) is a homobifunctional crosslinker,which reacts with CS via either a Schiff base reac-tion, leading to imine functionality, and/or throughMichel-type adducts with terminal aldehydes, leadingto the formation of carbonyl groups (figure 1i). GA’s dis-advantage is that it is cytotoxic in its unreacted form, afact that has lead to reservations towards GA-crosslinkedCS for biomedical applications and encouraged oursearch for alternative, more biocompatible crosslinkers.

Genipin (figure 1b) was first isolated from extractsof the Genipa americana plant; it is also found in low

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concentrations (,0.1%) in Gardenia jasminoides Ellisfruits. At present, genipin is isolated after hydrolysis ofgeniposide, from the gardenia plant, but in higher con-centrations (3.06–4.12%). The hydrolysis process usesb-glucosidase from Penicillium nigricans [28]. Becausegenipin is a naturally occurring crosslinker with 5000–10 000 times less cytotoxicity than GA [29], it haswidely been studied as an alternative crosslinker forCS-based biomaterials. Genipin spontaneously crosslinksCS gels, microspheres, films or cast membranes in0.5–5.0% acetic acid (AA). Most of the time, however,it is crosslinked in blends with synthetic polymers suchas PVA [30], poly (vinylpyrrolidone) (PVP) [31] andPEO [32] and with the biopolymers gelatin [33,34]and silk fibroin [35]. The mechanism of crosslinkingis the spontaneous reaction of genipin with the NH2

group of CS (figure 1a,b,f ) [24] or a protein with areactive amino-group; it forms dark blue or greenpigments [22–24].

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Another known CS crosslinker is hexamethylene-1,6-diaminocarboxysulphonate (HDACS; figure 1c). HDACS,a water-soluble and stable blocked-diisocyanate, formsurea linkages (figure 1g) after crosslinking with the NH2

group of CS at basic pH or at elevated temperatures[25,26]. Like genipin and HDACS, the epoxide, epichloro-hydrin (ECH) (figure 1d), can crosslink at the NH2 group,but has also been demonstrated to crosslink with primaryOH groups in CS films [36], beads [21,27] and wet-spun fibres [37–39] (figure 1h1,h2). The mechanism ofCS–ECH crosslinking is temperature-dependent [36].

To the best of our knowledge, to date only GA(figure 1e) has been successfully used to crosslink electro-spun CS fibres [18,19] (figure 1i). Explored in this studyis the use of a novel set of crosslinkers, genipin, HDACSand ECH, for the processing of crosslinked electrospunCS fibres and mats. Their effect on fibre and mat mor-phology and the chemical stability of as-spun andcrosslinked fibre mats are reported here. Their effect onthe mechanical performance of the mats is described inpart 2 of this publication series [40].

2. MATERIAL AND METHODS

2.1. Reagents

Medium molecular weight CS (75% DD, MW ¼ 190–310 kDa), TFA (99% ReagentPlus), GA (50 wt% inwater), ECH, AA (.99.7 þ % ACS Reagent) andsodium hydroxide (NaOH) were used as received fromSigma Aldrich, MO, USA. Genipin was purchased fromWako Pure Chemicals Industry, Ltd., (Japan). HDACSwas prepared using the protocol reported by Welshet al. [25]. All aqueous solutions were prepared withdoubly distilled water.

2.2. Preparation of chitosan solutions

CS solutions were prepared with 2.7 wt% CS in 99 per centTFA, mixing the solutions overnight at room temperatureon an Arma-Rotator A-1 (Bethesda, MD, USA). Solutionsof 2.7 wt% CS in 1 per cent TFA/water were also preparedfor conductivity measurements.

2.3. Crosslinking

The electrospinning and crosslinking procedures were amodification of the work of Schiffman and Schauer [19].The respective crosslinker was added to the CS/TFAsolution and mixed for 2 min immediately before elec-trospinning. The amount of crosslinkers added was asfollows: genipin (0.1 wt%); 5 : 1 (wt%) CS : HDACS[25,41]; 10 : 1 (vol%) CS : ECH [21,27,42]. For compari-son, as-spun mats were likewise prepared by adding1 ml of GA to a 2.7% (w/v) CS/TFA solution [19].

2.4. Viscosity, pH and conductivity of solutions

All viscosity measurements were performed on the samesolutions used for electrospinning. Methods were basedon the ASTM D446 standard method using an Ubbe-lohde glass capillary viscometer (Cannon InstrumentCo., State College, PA, USA).

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The pH and conductivity of the solutions prior tospinning were determined using universal range (pH0–14) pH indicator sticks (J.T. Baker, Deventer, TheNetherlands) and an Oakton CON 5110 conductivitymeter (Vernon Hills, IL, USA), respectively. All testswere performed at room temperature (25+ 48C) andwith three trials for each solution composition.

2.5. Electrospinning of crosslinked chitosansolutions

The CS/crosslinker solution was loaded into a syringe(Becton Dickinson & Co., Franklin Lakes, NJ, USA)to which a 21-gauge Precision Glide needle (BectonDickinson & Co.) was attached. The syringe was thenplaced on an advancement pump (Harvard Apparatus,Plymouth Meeting, PA, USA), set at 1.0 ml h–1 flowrate and a distance of 10 cm from the collecting plate,which was a 90 � 90 mm copper plate wrapped withaluminium foil. A high voltage supply (Gamma HighVoltage Research Inc., Ormond Beach, FL, USA) wasconnected to the needle (positive electrode) and theplate. A voltage of 15 kV was applied while advancingthe solution at a set flow rate. The set-up was runat 23–258C and 20–35% RH. All mats were stored atthe same conditions for a maximum of one week priorto conducting further tests.

2.6. Heat and base activation

As-spun and crosslinked samples underwent eitherheat or base activation. The respective temperaturesand thermal and base activation times are shown intable 1. Base activation was carried out in a 110 �80 � 50 mm gas vapour chamber (VWR Scientific Pro-ducts, Bridgeport, NJ, USA) containing 10 ml 1 MNaOH, which was allowed to vapourize at 238C. TheCS–GA and CS–genipin samples were exposed neitherto heat nor to base activation.

2.7. Field emission scanning electronmicroscopy

Fibre and mat morphologies of the electrospun sampleswere observed in Zeiss Supra 50VP field emission scan-ning electron microscopy (FESEM; Carl Zeiss NTS,LLC, North America) after sputter coating the sampleswith approximately 5 nm-thick platinum/palladium for30 s and 40 mA using Denton Vacuum Desk II (DentonVacuum, LLC, Moorestown, NJ, USA). Mean fibrediameters (n ¼ 50) were measured using IMAGEJ(v. 1.41o, National Institute of Health, USA).

2.8. Fourier transform infrared microscopy

Infrared spectra for CS fibres post-crosslinking weretaken using Fourier transform infrared microscopy(FTIR; Varian Excalibur FTS-3000, Varian, Inc.,Palo Alto, CA, USA). All spectra were taken usingthe attenuated total reflectance module in the spectralrange of 4000–500 cm21 by accumulation of 64 scansat 4 cm21 resolution.

Table 1. Thermal and base activation conditions, and the solution and mat colour of CS samples.

sample activation solution colour mat colour

CS/TFA none clear, light yellow white608C, 24 h clear, light yellow white1208C, 2 h clear, light yellow whiteNaOH, 24 h clear, light yellow white

CS–GA none light to dark yellow yellowCS–genipin none yellow to brownish red light pinkCS–HDACS none light yellow, turbid yellow

1208C, 2 h light yellow, turbid yellowNaOH, 24 h light yellow, turbid yellow

CS–ECH none light yellow white to glossy opaque608C, 24 h light yellow opaqueNaOH, 24 h light yellow opaque

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2.9. Solubility test

Stability of the fibre mats was tested in acidic, neutral andbasic conditions. A modified procedure of the solubilitytests described by Samuel et al. [43], Gotoh et al. [44] andPark et al. [45] was performed. Samples (10 � 10 mm)were submerged in separate solutions, each containing20 ml 1 M AA (pH 3), H2O (pH 7) and 1 M NaOH (pH13) in a 100 � 15 mm round glass Petri dish (BectonDickinson & Co.). Moreover, the dissolution of mats wasmonitored by measuring the transmittance of each solutionat600 nm(T600)usinga spectrometer (USB2000MiniatureFibre Optic Spectrometer, Ocean Optics, Inc., Dunedin,FL,USA).Aliquots (1.5 ml)were taken, transferred to cuv-ettes andmeasured at an ambient temperature after 15 minand 72 h. Three trials were conducted for each measure-ment. The same volume reagent (1.5 ml) was replacedinto the solution after every sampling. Each mat conditionwas also visually observed and noted. For reference values,1 M AA, H2O and 1 M NaOH were used, and all thecompositions were normalized with respect to the corre-sponding reference solution (i.e. 1 M AA) rather than thereference-crosslinker (i.e. 1 M AA þ genipin) solutions.

2.10. Thermogravimetric analysis

Thermogravimetric analysis (TGA) was carried outwith a TGA Q50 Thermogravimetric Analyzer (TAInstruments, New Castle, DE, USA). The sampleswere placed in a humidity chamber at 65 per cent RHfor 1 h before testing. Samples of 3 mg from each com-position and treatment were individually loaded into aplatinum pan and examined to determine the waterloss difference between the fibre mats. Runs were car-ried out under N2 gas and with a ramp rate of58C min21 up to 1508C. The per cent weight loss (%wt. loss) was normalized per milligram (mg) of the mat.

3. RESULTS AND DISCUSSION

3.1. Solution properties, electrospinning andfibre morphology

3.1.1. Solution colour, mat colour and pre-activationfibre morphologyAll CS solutions prepared in this study were electrospin-nable. Owing to the corrosiveness of the TFA solutions,

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viscosities were taken using the Ubbelohde glass visc-ometer. Viscosities did not drastically change thespinning process (3 h) (figure 2m); however, it is impor-tant to note that, as expected, the relative viscosity ofCS/TFA increased after the addition of genipin andECH. The addition of HDACS, however, decreasedthe solution viscosity and also led to the formation ofa cloudy white dispersion that phase separates throughtime, requiring the constant replacement and mixing ofa freshly prepared solution into the syringe every hour.Owing to this, CS–HDACS solutions were filtered priorto measurement of viscosity to prevent clogging thecapillary tube.

Mat characteristics varied depending on the crosslin-ker added and/or the activation conditions used.Table 1 shows the observed solution and fibre mat col-ours. It is important to note that initially, all CS/TFAsolutions were clear, light yellow in colour.

Genipin-crosslinked fibre mats were light pink incolour with both unbranched round and flat fibres.This is in contrast to observed dark blue or greencolour that is observed when genipin is added to CS/AA in films or beads [24]; the addition of genipin toCS/TFA changed the colour to a darker yellow. As elec-trospinning progressed, the solution turned brownishred. Yellow and brownish-red intermediate pigmentshad been observed previously when genipin was addedto methylamine [24]. Researchers have noticed thatfurther exposure of the pigments to O2 leads to the for-mation of a blue pigment [22,23]. We also noticed thatthe CS–genipin mats turn blue when placed in aNaOH–ethanol solution.

Genipin’s degree of crosslinking with CS is pH-dependent [24]. At a higher pH, a higher degree ofself-polymerization is likely to occur than at lower pH.This self-polymerization occurs before genipin reactswith CS, leading to a lower degree of crosslinking.Genipin-crosslinked CS networks at a lower pH consistof primary CS chains and short crosslink bridges of geni-pin, while higher pH leads to networks with long crosslinkbridges of genipin [24]. Because the spinning solution is atpH � 2 (figure 2o), the brownish red colour of the sol-ution is probably owing to short crosslink bridges ofgenipin when crosslinking to the NH2 group on CS.

Liu et al. [46] reported that to fully crosslink CS,0.025 wt% genipin is sufficient. In this study however,

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Figure 2. Scanning electron microscope micrographs of electrospun fibres: (a) CS, (b) CS-608C, (c) CS-1208C, (d) CS-base, (e)CS–GA, ( f ) CS–genipin, (g) CS–HDACS, (h) CS–HDACS-1208C, (i) CS–HDACS-base, ( j) CS–ECH, (k) CS–ECH-608Cand (l ) CS–ECH-base. The plots of the: (m) change in relative viscosities through spinning time (blue downward triangles,CS/TFA; red squares, CS–genipin; green upward triangles, CS–HDACS; purple crosses, CS–ECH); fibre diameter effects asa function of solution, (n) relative viscosity prior to spinning, (o) pH and ( p) conductivity. (n–p) Red squares denote fibrediameter; green diamonds. (Online version in colour.)

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0.10 wt% was chosen for two reasons. First, excess of thecrosslinker was added to saturate all the crosslinking sitesof CS. Second, at concentrations less than 0.10 wt%, themats were soluble in acidic and neutral solutions, indicat-ing that the mats were not fully crosslinked. The0.10 wt% used had shown improvements in stability, aswill be briefly described later in this paper.

HDACS crosslinking produced white fibre mats withboth round and flat non-uniform diameters. Addition ofHDACS made the CS/TFA solution turbid without anobserved colour change. Amounts added for bothHDACS [25,26] and ECH [21,27,42] were based on pre-vious studies on CS films or hydrogels. Keeping in mindthat the CS used is 75 per cent DD, the crosslinkeramounts used here were modified to estimate a 1 : 1molar ratio of amine : crosslinker group. ECH-crosslinkedfibre mats were initially, after 10 min of electrospinning,transparent and glossy, but turned white after 5 h.Fibres were round, highly branched and tree-root-like.

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3.1.2. Pre-activation fibre diameterFigure 2a–l displays the fibre surface morphologies ofall the spun mats. Electrospinning the CS/TFA sol-ution yields a mean fibre diameter of 133+ 53 nm.When adding the crosslinker genipin, HDACS andECH, the mean fibre diameters increased. The CS–gen-ipin, CS–HDACS and CS–ECH mats consisted offibres with mean fibre diameters of 267+ 199 nm,644+ 359 nm and 896+ 435 nm, respectively. Forcomparison, CS–GA crosslinked mats were also spun.The yellow-coloured CS–GA mats have a mean fibrediameter of 112+ 33 nm, which is lower than that ofthe other three tested crosslinkers. This value is lowerbut within the standard deviation of the earlierreported value of 128+ 40 nm [18,19].

The increase in fibre diameters upon addition of thecrosslinker to the CS solution is probably owing toseveral factors. In the case ofCS–genipin, thefibre increasewas very minimal, indicated by minimal change in

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viscosity (figure 2m) or conductivity (figure 2p) of the sol-ution. However, it is suggested that fibre diameter increaseis probably owing to the slow formation of short crosslinkswithin the polymer chains at low pH (figure 2o), prevent-ing thinning of the strand. In ECH, since the crosslinkerdoes not ionize in solution (figure 2o,p) pH and con-ductivity changes were not substantial. However, themarked increase in diameters might be owing to initialcrosslinker modification of the CS backbone (i.e. a bifunc-tional crosslinker binding to only one CS functional groupinstead of two; figure 1) and/or formation of stronger poly-mer chain with crosslinker bonds (i.e. increased formationof covalent bonds that increase polymer chain rigidity),supported by the increased relative viscosities of the sol-ution prior to spinning (figure 2n), all of which preventthinning of the strand during fibre formation.

CS–HDACS exhibited an opposite behaviour to ECH,but it is important to remember that unlike the solutionsused for viscometry tests, the spinning solution was con-stantly replenished, used unfiltered and ionized insolution, leading to increase in conductivity (figure 2p).

Moreover, the strong acid and solvent TFA (pH 1–2,figure 2o) used in this study protonates the aminegroups (pKa 6.3), leading to a highly charged backbone(i.e. the uncharged amines become charged), whichincreases chain repulsion and/or the swelling capabilityof the fibres [47]. Owing to the differences in the chem-istries of the added crosslinkers, the presence andswelling effect of residual TFA in the mat itself maybeexpected to vary from one crosslinker type to another.This is supported by TGA investigations (figure 3a)in which smaller fibre diameters correlates to higherweight loss owing to surface adsorbed water.

3.1.3. Post-activation fibre morphology and diameterFull crosslinking of CS with HDACS [25,26] or ECH[21,27,36–39] requires either heat (1208C and 608C,respectively) or base activation. Figure 2 shows scan-ning electron microscope (SEM) micrographs of thepost-activation mats.

For CS–HDACS-1208C, mean fibre diameters were285+ 139 nm, a 55 per cent decrease from the as-spun CS–HDACS. Like heat activation, exposure ofCS–HDACS-base to 1 M NaOH for 24 h showed adecrease in mean fibre diameter to 339+ 179 nm, a 47per cent decrease. In both activation conditions, nochanges in mat colour and shape were observed.A decrease in mean fibre diameters was also observedfor CS–ECH-608C (870+ 490 nm), a 3 per centdecrease, and CS–ECH-base (394+ 263 nm), a 56 percent decrease. In both cases, the mats changed fromglossy/white to glossy/rough/white in texture.

The decrease in fibre diameters maybe attributed to theloss of water from the fibre mats brought about bythe increased temperature and/or the full crosslinking athigher pH. This is also supported by the per cent weightloss (figure 3a) as determined by TGA, which indicatethat mats with lower fibre diameters had exhibitedhigher water loss, which was also expected because ofthe surface-adsorbed water. It must be noted that all thespun mats have been conditioned at 65 per cent RH priorto running TGA to ensure that the effects are only owingto the crosslinker and/or activation conditions. The

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minimal decrease in fibre diameter for CS–ECH-608Cmight be owing to the incomplete evaporation of water asa result of lower activation temperature or because ECHcrosslinking is more pH- [38] than temperature-mediated.

The comparison of the mean fibre diameters of the as-spun with the pre- and post-activated crosslinked CS mats(figure 2) revealed that in the case of the as-spun mat(CS), both the heat and the base treatments (CS-608C,CS-1208C and CS-base) resulted in an increase in meanfibre diameters. Interestingly, the opposite, a decreasein fibre diameter, was observed in the case of thepre-activation samples (CS–HDACS and CS–ECH)after crosslinker activation with heat or base (CS–HDACS-1208C, CS–HDACS-base, CS–ECH-608C andCS–ECH-base) owing to chemical crosslinking.

3.2. Fourier transform infrared microscopy

FTIR spectra (figure 3) of as-spun and crosslinked fibremats were taken to determine their respective chemicalinteractions, especially any expected covalent cross-linking. Characteristic CS peaks (figure 3) are fromamide I (1673 cm21), amide II (1532 cm21), C–Nstretch (1431 cm21), bridge ether oxygen (1202 cm21)and alcohol C–O (1085 cm21) [18,19].

With the addition of crosslinkers, changes in CS IRpeaks were observed. CS–genipin (figure 3) exhibitedamide I peak broadening, which can be attributed toNH2 group deformation [24]. The reaction mechanism ofcrosslinking of genipin with CS is chemically complexand pH-dependent [22–24]. Under acidic and neutralpH, genipin crosslinking involves the attack of the NH2

of CS, resulting in a possible formation of bifunctional lin-kages to the crosslinker [24,48] (figure 1f ).

HDACScrosslinksCSat the NH2 group of the polymer,creating a urea linkage [25] (figure 1g). When fully cross-linked, this appears as a strong peak at around1650 cm21 [25]. In this study, a medium peak at around1652 cm21 (figure 3) was evident for the CS–HDACSfibres even before heat (figure 3) or base exposure(figure 3), indicating partial covalent crosslinking. Thesame peak increased in height after heat (figure 3) orbase exposure (figure 3), indicating increased crosslinking.

ECH is another known CS crosslinker, which isfavoured owing to its ability to couple the polymer atthe OH group, leaving more chemical functionality tothe polymer owing to the available NH2 groups. However,crosslinking is not limited to the OH groups (figure 1h1),but also occurs at the NH2 groups (figure 1h2) dependingon the temperature [36]. At less than 408C, ECH cross-links CS at the NH2 groups but at more than 408C,both the OH and the NH2 groups participate (figure1h1,h2), forming a denser crosslinked network [36].

The FTIR spectrum (figure 3) of CS–ECH matsshow amine deformation at 1638 cm21 and an increasein C–N stretch peak (1431 cm21) suggesting cross-linking occurs at the NH2 groups of CS. These peakswere also taller and broader after heat or base exposureindicating an increase in amine group deformation,which might be used in crosslinking. Moreover, thepeak at 1085 cm21 (figure 3) that corresponds to theC–O stretch was observed to increase and broaden.This is an indication that the OH groups are

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Figure 3. (a) Weight loss (%, red squares) and mean fibre diameters (blue diamonds) of CS-based fibres. FTIR spectra ofCS-based mats: (b) CS; (c) CS–genipin; (d) CS–HDACS; (e) CS–HDACS-1208C; ( f ) CS–HDACS-base; (g) CS–ECH;(h) CS–ECH-608C; and (i) CS–ECH-base; broken vertical lines correspond to (1673) amide I; (1638) amine deformation;(1532) amide II; (1431) C–N stretch; (1202) bridge C–O–C; (1085) alcohol C–O stretch. Downward arrow at 1652 indicatespossible urea linkage. (Online version in colour.)

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crosslinking at elevated temperatures, even under basicconditions (figure 3).

3.3. Solubility test

Crosslinking improves the dissolution of CS films, beadsand fibre mats [18,19] under a wide pH range. Figure 4

J. R. Soc. Interface

and table 2 summarizes the results of the dissolutiontest. As dissolution criteria, transmittances at 90 [43]and 50 [45] were taken as cutoffs (figure 4). If the trans-mittance (T600) of the aliquot solutions from theimmersed mats is greater than 50, the mats were con-sidered partially dissolved. Further, if the T600 isgreater than 90 and the mat is visually present in the

0

30

60

90

120

CS

CS-bas

e

CS–gen

ipin

CS–HDACS

CS–HDACS-b

ase

CS–HDACS-1

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CS–ECH-6

0°C

CS-60°

C

CS-120

°C

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ase

CS–GA

refere

nce

T60

0

1M NaOH (pH 13)

0

30

60

90

120

T60

0

1M acetic acid (pH 3)

0

30

60

90

120

T60

0

H2O (pH 7)

Figure 4. Transmittance (T600) of aliquot solutions from dissolution testing of CS-based mats in 1 M AA, H2O and 1 M NaOH for15 min (unfilled bars) and 72 h (blue bars). Solid horizontal bars indicate T600¼ 90, while broken line mark T600¼ 50. (Onlineversion in colour.)

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solution, it is considered not dissolved; otherwise, it isfully dissolved (figure 4 and table 2).

Knowing that CS (figure 5d) does not dissolve under1 M NaOH owing to the neutralization of the aminegroups of CS, we are interested in knowing which cross-linker and/or activation conditions are able to keepthe fibrous structures of the mats after immersionin solutions with the lowest pH at a longer time.Figure 5 displays the SEM micrographs of these mats.At neutral to acidic pH, CS is soluble owing to the pro-tonation of amines (pKa 6.3), unless the polymer iscrosslinked or the functional groups are modified.Hence, improved mats are those that are stable atlower pHs at a longer time. Here, like as-spun CS,

J. R. Soc. Interface

CS-608C (figure 5h) is stable only at 1 M NaOH. Heat-ing of CS (CS-1208C, figure 5k) mats partially improvedstability up to 15 min at pH 3, which is attributed tothe modification of the amine groups by the formationof an amide between the ammonium salt and the tri-fluoroacetate at temperatures greater than 1008C [49].CS-base (figure 5j) is only partially stable for 15 minat pH 3.

Improved stability of the mats was observed uponthe addition of the crosslinkers. Addition of genipin ren-dered the mats partially insoluble at pH 3 even after72 h (figure 5g), supporting the suggestion that genipinformed partial crosslinks during spinning. Interestinglyand as expected, the addition of HDACS rendered the

Table 2. Transmittance (T600) of aliquot solutions from dissolution testing of CS-based mats in 1 M AA, H2O and 1 M NaOHfor 15 min and 72 h with the visual observations of mat solubility. (Visual observations of mat solubility indicate N and Y fornot visible and visible, respectively.)

mats

15 min 72 h

visual observation T600 comments visual observation T600 comments

1 M AA pH 3CS N 95 fully dissolved N 113 fully dissolvedCS-608C N 84 fully dissolved N 111 fully dissolvedCS-1208C Y 86 partially dissolved N 101 fully dissolvedCS-base Y 83 partially dissolved N 93 fully dissolvedCS–genipin Y 80 partially dissolved Y 86 partially dissolvedCS–HDACS Y 94 not dissolved Y 105 not dissolvedCS–HDACS-1208C Y 93 not dissolved Y 104 not dissolvedCS–HDACS-base Y 105 not dissolved Y 114 not dissolvedCS–ECH N 86 fully dissolved N 94 fully dissolvedCS–ECH-608C Y 56 partially dissolved N 88 fully dissolvedCS–ECH-base Y 88 partially dissolved Y 105 not dissolvedCS–GA Y 87 partially dissolved Y 103 not dissolved1 M AA (reference) — 98 — — 93 —

H2O pH 6-7CS N 108 fully dissolved N 79 fully dissolvedCS-608C N 108 fully dissolved N 91 fully dissolvedCS-1208C Y 100 not dissolved Y 80 partially dissolvedCS-base N 101 fully dissolved N 103 fully dissolvedCS–genipin Y 95 not dissolved Y 86 partially dissolvedCS–HDACS Y 93 not dissolved Y 92 not dissolvedCS–HDACS-1208C Y 110 not dissolved Y 91 not dissolvedCS–HDACS-base Y 95 not dissolved Y 91 not dissolvedCS–ECH Y 69 partially dissolved N 60 fully dissolvedCS–ECH-608C Y 86 partially dissolved Y 60 partially dissolvedCS–ECH-base Y 99 not dissolved Y 81 partially dissolvedCS–GA Y 103 not dissolved Y 90 not dissolvedH2O (reference) — 98 — — 97 —

1 M NaOH pH 13CS Y 108 not dissolved Y 123 not dissolvedCS-608C Y 108 not dissolved Y 123 not dissolvedCS-1208C Y 112 not dissolved Y 120 not dissolvedCS-base Y 112 not dissolved Y 127 not dissolvedCS–genipin Y 113 not dissolved Y 110 not dissolvedCS–HDACS Y 120 not dissolved Y 133 not dissolvedCS–HDACS-1208C Y 121 not dissolved Y 120 not dissolvedCS–HDACS-base Y 118 not dissolved Y 114 not dissolvedCS–ECH Y 118 not dissolved Y 133 not dissolvedCS–ECH-608C Y 120 not dissolved Y 111 not dissolvedCS–ECH-base Y 122 not dissolved Y 116 not dissolvedCS–GA Y 101 not dissolved Y 121 not dissolved1 M NaOH (reference) — 99 — — 103 —

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mats fully insoluble after 72 h (figure 5a–c). This iscomparable to CS–GA (figure 5f ).

In the case of ECH addition, the improved matswere partially stable at neutral pH for a short time(figure 5l ). Although chemical interactions indicatingpossible participation of hydroxyl groups at elevatedtemperatures for the CS–ECH-608C (figures 3c and 5i)were observed, base activation resulted in a more stablemat (CS–ECH-base, figure 5e) that did not dissolveeven after 72 h at pH 3.

Fibre morphologies of the surviving mats were simi-lar to the pre-dissolution test samples. Moreover, thefibre diameters of the mats (figure 5) after the dissol-ution tests are still within the standard deviation ofthe initial mats.

J. R. Soc. Interface

Overall, the addition of crosslinker and/or post-activation steps resulted in mats with better dissolution,retained fibrous morphologies and mean fibre diametersin comparison to their corresponding controls (as-spunCS, CS-608C, CS-1208C or CS-base; figure 5).

4. CONCLUSION

Novel sets of crosslinkers for electrospun CS fibre matswere investigated using the three crosslinkers genipin,HDACS and ECH. Both the type of crosslinker andpost-spinning activation through exposure to tempera-tures of 608C and 1208C or a base affected the fibrediameters and mat morphology. FTIR spectra revealed

time pH of solutions that the mats were exposed to

3 6 – 7 13

72 h none observed

15 min none observed

(a) (b)

(e) (f ) (h)

(c) (d)

(g*)

(i*) (j*) (k*) (l*)

1 µm

1 µm

2 µm 2 µm 2 µm 2 µm

2 µm

2 µm2 µm1 µm

1 µm1 µm1 µm

Figure 5. Selected micrographs of electrospun CS-based mats after dissolution test. Images display the lowest pH and longest timethat the mats were able to retain their fibrous structure after exposure to all the conditions. Compositions with an asterisk (*) arepartially dissolved while those without did not dissolve: (a) CS–HDACS-1208C (434+326 nm), (b) CS–HDACS-base (228+163 nm), (c) CS–HDACS (389+ 267 nm), (d) CS (261+107 nm), (e) CS–ECH-base (69+19 nm), ( f ) CS–GA (103+44 nm), (g*) CS–genipin (176+ 106 nm), (h) CS-608C (519+225 nm), (i*) CS–ECH-608C (498+ 441 nm), ( j*) CS-base(146+109 nm), (k*) CS-1208C (149+ 136 nm) and (l*) CS–ECH (410+163 nm).

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covalent chemical interactions of CS with genipin,HDACS and ECH. Activation conditions improvedthe mat stability at a wider pH range and longertime. These findings are substantial as it extends thepotential applications of CS-based electrospun fibresand allows ease in determining the appropriate crosslin-ker and/or activation protocols required to fabricatemats for more specific applications and conditionssuch as in biomedical engineering. The effect of thedifferent crosslinkers and post-spinning treatments onthe mechanical properties of the mats are described inpart 2 of this publication series.

The authors thank: Dr Giuseppe Palmese for use of his FTIRand Aldo DiPrato and Amy Peterson for helping with theFTIR; Keith Fahnestock for help with TGA; and Dr EdBasgall of the Centralized Research Facilities (CRF),College of Engineering, Drexel University for use of FESEM.M.S.A. thanks the Institute of Food Technology (PAsection) and Drexel University Freshman DesignEngineering Fellowship. A.E.D. thanks the PhiladelphiaSWE for the Dow Chemical Company Award andacknowledges her GAANN fellowship P200A070496 andNSF-IGERT 0654313. U.G.K.W. thanks Anne Stevens forthe generous support of her research and group. Theauthors acknowledge funding by the NSF DMR grant no.0907572, NSF CMMI grant no. 0804543 and Ben FranklinNanotechnology Institute, Philadelphia, PA, USA.

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