56 Chiang Mai J. Sci. 2011; 38(1)

Electrospun Zein Fibrous Membranes UsingGlyoxal as Cross-Linking Agent: Preparation,Characterization and Potential for Use in BiomedicalApplicationsOrawan Suwantong*[a], Prasit Pavasant [b] and Pitt Supaphol**[c,d][a] School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand.[b] Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand.[c] The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand.[d] The Center for Petroleum, Petrochemical and Advanced Materials, Bangkok 10330, Thailand.Author for correspondence; e-mail: * o.suwantong@gmail.com, ** pitt.s@chula.ac.th

Recieved: 25 April 2010Accepted: 5 July 2010

ABSTRACTIn the present contribution, ultra-fine zein fibers were successfully prepared

by electrospinning. The diameters of these fibers ranged between 0.32 and 1.10 μm.Cross-linked electrospun zein fiber mats were prepared by adding varying quantities of glyoxalaqueous solution (i.e., 0, 5, 10 and 15% v/v based on the volume of the zein solution) into thebase zein solution (i.e, 40% w/v). Mechanical properties of the electrospun zein fiber matswere evidently improved by the cross-linking with glyoxal. The fibrous nature of the cross-linked materials was preserved even after submersion in distilled water at room temperature(i.e., 25 1oC) or 37oC for 5 d. The potential for use of the cross-linked electrospun zein fibermats as scaffolding materials for cell/tissue culture was assessed with indirect cytotoxicityevaluation with mouse fibroblasts (L929) and direct culture with human foreskin fibroblasts(HFF). It was found that an increase in the amount of glyoxal used to cross-link the materialscaused the cross-linked materials to be more toxic, but this can be alleviated by pre-treatmentof the cross-linked electrospun zein fiber mats in a glycine aqueous solution. Finally, the cultureof HFF on some of the cross-linked fiber mat samples for 5 d showed that the cells grewwell on the materials.

Keywords: electrospinning, zein, fibrous scaffolds.

1. INTRODUCTIONElectrospinning is an established method

for producing fibrous membranes withdiameters of the underlying fibers ranging

from less than ten micrometers down to tensof nanometers [1]. The principle of the processinvolves the application of a strong electric

Chiang Mai J. Sci. 2011; 38(1) : 56-70www.science.cmu.ac.th/journal-science/josci.htmlContributed Paper

Chiang Mai J. Sci. 2011; 38(1) 57

field across a finite distance between a conductivecapillary, connected to a reservoir of a polymersolution, and a collecting device. Under a highenough electric field, the partially-sphericalshape of a pendant droplet of the polymersolution at the capillary tip is deformed into aconical shape. If the electric field exceeds athreshold value, electrical forces overcomethat of the surface tension, resulting in anejection of the polymer solution. The ejected,charged stream of the polymer solution (i.e.,charged jet) moves towards the collectingdevice, resulting in the deposition of ultra-thin polymeric fibers on the collecting deviceas a non-woven membrane [1]. Parametersaffecting the morphology of the fibers are,for examples, solution properties (e.g., viscosity,conductivity, surface tension, etc.), solutionfeed rate and electric field [2,3]. Due to thesimplicity of the technique and the uniquecharacteristics of the obtained fibers, thesefibrous materials have been heavily exploredfor various biomedical applications, includingwound dressings [4,5], carriers for drugdelivery [6-8], and scaffolding materials forcell/tissue culture [9-13].

Zein is a major storage protein, makingup to about 45-50% of the total proteinsfound in maize. The molecular structure ofzein is helical wheel conformation in whichnine homologous repeating units are arrangedin an anti-parallel manner which is stabilizedby hydrogen bonds [14]. Zein exhibits variousinteresting properties, such as toughness,flexibility, compressibility, glossiness,hydrophobicity, resistance to microbial attack[15] and antioxidant activity [16-18]. Theproposed uses of zein in biomedicalapplications are as carriers for drug delivery[19-21], food packaging [22] and scaffoldingmaterials for cell/tissue culture [23,24].Miyoshi et al. [25] were the first group to reportsuccessful electrospinning of zein from itssolutions in 80% w/w ethanol aqueous

solution. Yao et al. [26] reported successfulelectrospinning of zein from its solutionsin 70-90% v/v ethanol aqueous solutions.A more thorough study on the electrospinningof zein from its solutions in various ethanol-based solutions was reported by Torres-Gineret al. [27], who investigated a number ofparameters (i.e., polymer concentration,solvent content, solution flow-rate, appliedpotential, needle tip-to-collector distance andpH) that affected the electrospinning of thepolymer. Cross-linking of electrospun zeinfiber mats from zein solutions in variousethanol aqueous solutions with hexamethylenediisocyanate (HDI) was reported by Yao etal. [26] Selling et al. [28] used glutaraldehydeas the cross-linking reagent to improve thephysical properties and solvent resistance ofelectrospun zein fiber mats. The use of citricacid as the cross-linking reagent for electrospunzein fiber mats and sodium hypophosphitemonohydrate as the catalyst was reported byXu et al. [29]

In the present contribution, ultra-fine zeinfiber mats were prepared by electrospinning.The effects of solution concentration, appliedelectrical potential and collection distance onmorphological appearance of the obtainedfibers and on the size of the individual fibersegments were investigated by scanningelectron microscopy (SEM). To improvestability and mechanical integrity of the fibermats in an aqueous medium, the electrospunzein fiber mats were cross-linked with glyoxal.The potential for use of the neat and the cross-linked zein fiber mats as fibrous membranesfor various biomedical applications, such aswound dressings, was evaluated with murinefibroblasts (L929) and human foreskinfibroblasts (HFF).

2. EXPERIMENTAL DETAILS2.1 Materials

Zein powder (Mr = 25,000-29,000 g⋅mol-1)

58 Chiang Mai J. Sci. 2011; 38(1)

was purchased from Fluka Chemika/Biochemika (Switzerland). Ethanol (80% v/vaqueous solution; Carlo Erba, Italy) was usedas the solvent. Glyoxal (40% v/v aqueoussolution; Fluka Chemika/Biochemika,Switzerland) was used as the cross-linkingagent. All other chemicals were of analyticalreagent grade and used as received.

2.2 Preparation of Neat and Cross-LinkedZein Fiber Mats

To prepare the neat electrospun zein fibermats, zein solutions in the aqueous ethanolsolution were prepared at the concentrationsof 30 to 45% w/v. Prior to electrospinning,the as-prepared spinning solutions werecharacterized for their viscosity andconductivity using a Brookfield DV-IIIprogrammable viscometer and a SUNTEXconductivity meter, respectively. Each of theas-prepared spinning solutions was latercontained in a 5-mL glass syringe, the openend of which was connected to a blunt20-gauge stainless steel hypodermic needle(OD = 0.91 mm), used as the nozzle. AGamma High Voltage Research D-ES30PN/M692 power supply was used to generate aDC electrical potential in the range of 15 to25 kV. The electrical potential of positivepolarity was supplied to the solution across afinite distance between the tip of the needleand a sheet of Al foil wrapped around astationary plastic backing, used as the collectingdevice, ranging between 10 and 20 cm (i.e.,collection distance). The solution feed rate wasmaintained by a syringe pump at 1.5 mL⋅h-1

and the collection time was fixed at about5 min.

To prepare the cross-linked electrospunzein fiber mats, the 40% w/v zein solution inthe ethanol aqueous solution was mixed withvarying amounts of the glyoxal aqueoussolution (i.e., 0, 5, 10 and 15% v/v based onthe volume of the zein solution). Prior to

electrospinning, each of the spinning solutionswas characterized for their viscosity andconductivity using the viscometer and theconductivity meter, respectively. Electro-spinning of the as-prepared zein solutionswith or without the presence of the glyoxalaqueous solution was carried out at a fixedelectrical potential of 20 kV over a fixedcollection distance of 15 cm onto a sheet ofAl foil wrapped around a home-maderotating cylinder (rotational speed ≈ 40 rpm),used as the collecting device. The solution feedrate was also fixed at 1.5 mL⋅h-1 and thecollection time was fixed at about 20 h. Theelectrospun fiber mats were dried in an ovenat 60oC for 12 h. Hereafter, ZP, ZP5, ZP10and ZP15 are used to refer to the electrospunzein fiber mats that had been fabricatedfrom the zein solutions containing 0, 5, 10 and15% v/v of the glyoxal aqueous solution,respectively.

2.3 Characterization of Neat and Cross-Linked Zein Fiber Mats

Morphological appearance of both theneat and the cross-linked electrospun zeinfiber mats was observed by a JEOL JSM-6400 scanning electron microscope (SEM).The fiber mat specimens were coated with athin layer of gold prior to SEM observation.Diameters of the individual fiber segmentswere measured directly from the SEM imagesusing a SemAphore 4.0 software, with theaverage values being calculated from at least100 measurements. In the cases where beadedfibers were obtained, the average number ofbeads per unit area (i.e., the bead density) andthe average diameters (i.e., only measured onthe fiber segments between beads) of thebeaded fibers were calculated from measure-ments on SEM images of 3,500x and 1,000xmagnifications, respectively.

Mechanical properties in terms of thetensile strength, Young’s modulus and elongation

Chiang Mai J. Sci. 2011; 38(1) 59

at break of both the neat and the cross-linkedelectrospun zein fiber mats were investigatedusing a Lloyd LRX universal testing machine(gauge length = 50 mm and crosshead speed= 20 mm⋅min-1). The fiber mats of about70 10 mm in thickness were cut intorectangular-shaped specimens (10 mm × 100mm). For each group of fiber mat samples,about 5 specimens were tested and the resultswere reported as average values.

To assess the stability of the fiber matsin maintaining their fibrous structure aftersubmersion in an aqueous medium, both theneat and the cross-linked electrospun zein fibermat specimens (cut into circular discs of about1.4 cm in diameter) were submerged indistilled water at either room temperature(i.e., 25 1oC) or 37oC for either 1 or 5 d. Ata given time point, the morphology of thedried specimens was observed by SEM.

2.4 Cytotoxicity EvaluationIndirect cytoxicity evaluation of both the

neat and the cross-linked electrospun zein fibermats was conducted in adaptation from theISO 10993-5 standard test method, usingmouse fibroblasts (L929). Both the neat andthe cross-linked electrospun fiber mats werecut into circular discs (about 23 mm in diameterand about 60 5 mm in thickness). Thesespecimens were divided into two groups. Thespecimens in the first group were tested intheir native form, while those in the secondgroup were tested after they had been treatedwith 0.1 M glycine aqueous solution. Prior tofurther investigation, the specimens weresterilized with UV radiation for about 1 h.Extraction media were then prepared byimmersing the disc specimens in a serum-freemedium [SFM; containing Dulbecco’smodified Eagle’s medium (DMEM), 1%L-glutamine, 1% lactabumin and 1% antibioticand antimycotic formulation (containingpenicillin G sodium, streptomycin sulfate, and

amphotericin B)] in wells of a 24-well tissue-culture polystyrene plate (TCPS; BiokomSystems, Poland) for either 3 or 7 d. L929were seeded at 40,000 cells/well in serum-containing DMEM for 16 h to allow cellattachment. The cells were then starved withSFM for 24 h, after which time the mediumwas replaced with an extraction medium.The cells of the control group were incubatedwith fresh SFM. After 24 h, the viability ofthe cells was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay (see Supplementaryinformation).

2.5 Morphological Observation of Culturedcells

Human foreskin fibroblasts (HFF; at thedensity of 20,000 cells/well) were culturedon the cross-linked electrospun zein fiber mats(i.e., ZP10) at 37oC. After 5 d of cell culturing,morphological appearance of the cells wasobserved by SEM. After removal of theculture medium, the cell-cultured specimenswere rinsed with PBS twice and the cells werethen fixed with 3% gluteraldehyde solution[diluted from 50% glutaraldehyde solution(Electron Microscopy Science, USA) withPBS] at 500 μL/well. After 30 min, they wererinsed again with and kept in PBS at 4oC.After the cell fixation, the specimens weredehydrated in graded ethanol solutions(i.e., 30, 50, 70 and 90%, respectively) and inpure ethanol for about 2 min at eachconcentration. The specimens were thendried in 100% hexamethyldisilazane (HMDS;Sigma, USA) for 5 min and later in air afterthe removal of HMDS. After completelydried, the specimens were mounted on SEMstubs, coated with gold, and observed bySEM.

2.6 Statistical AnalysisData were presented as means ± standard

60 Chiang Mai J. Sci. 2011; 38(1)

errors of means (n = 3). A one-way ANOVAwas used to compare the means of differentdata sets, and statistical significance wasaccepted at a 0.05 confidence level.

3. RESULTS AND DISCUSSIONAs mentioned, Miyoshi et al. [25] were

the first group to report successful electro-spinning of zein from its solutions in 80%w/w ethanol aqueous solution. The effectsof the concentration of the zein solutions(i.e., 18-25 wt.%) and the applied potential(i.e., 15 and 30 kV over a fixed collectiondistance of 10 cm) on morphology and sizeof the electrospun zein fibers were reported.At 15 kV, electrospinning of the zein solutionswith concentrations greater than about21 wt.% produced smooth and ribbon-likefibers. At 30 kV, predominantly smooth andribbon-like fibers were obtained when theconcen-tration of the zein solutions wasgreater than about 18 wt.%. The diametersof the obtained fiber segments were reportedto be about 1 μm and 700 nm, respectively[25]. Yao et al. [26] studied the electrospinningof zein from its solutions in various ethanolaqueous solutions. The effects of the concen-tration of the zein solutions (i.e., 10-50% w/v)and the ethanol/water ratio of the ethanolaqueous solutions (i.e., 70:30, 80:20, and 90:10v/v) on morphology and size of the electro-spun zein fibers were reported. At 70:30 v/vof the ethanol aqueous solution, smooth andribbon-like fibers were obtained from 30-50% w/v zein solutions, with the diametersof these fibers ranging between about 1 and6 μm. It was also observed that increasing theethanol content in the solvent system causedthe resulting fibers to be more brittle, likely aresult of the self-assembling of the proteinduring solvent evaporation [26].

Recently, Torres-Giner et al. [27] studiedvarious parameters that affected the morphologyand size of the electrospun zein fibers. These

parameters are solution concentration, ethanolcontent in the ethanol aqueous solution usedas the solvent system, solution flow-rate,applied potential and collection distance. Theyfound that fibers were formed over theconcentration range of 25-50 wt.%. Duringthe initial increase in the concentration ofthe zein solutions between 25 and 33 wt.%,the change in the fiber diameters was low, withthe values being about 200 nm on average.Further increase in the solution concentrationto 42 and 50 wt.% resulted in a dramaticincrease in the fiber diameters, with the finalvalues being about 1 μm on average. Withregards to the effects of the ethanol content,the solution flow-rate and the appliedpotential, an initial increase in these parameters(i.e., 50-80% w/w for the ethanol content,0.1-0.46 mL⋅h-1 for the flow-rate and 7-11kV for the applied potential) did not have astrong influence on the diameters of theobtained fibers. Further increase in the valuesof these parameters resulted in an observedincrease in the diameters of the obtained fibers.Similarly, an initial increase in the collectiondistance in the range of 5-12.5 cm did nothave a strong effect on the diameters of theobtained fibers. Further increase in the valuesof this parameter, however, resulted in anobserved decrease in the diameters of theobtained fibers [27].

Prior to electrospinning, certain propertiessuch as shear viscosity and electrical conduc-tivity of the zein spinning solutions weremeasured and the results are summarized inTable 1. Here, the concentration of the ethanolaqueous solution used as the solvent systemfor zein was fixed at 80% v/v. According toTable 1, an increase in the concentration ofthe zein solutions from 30 to 45% w/v resultedin a monotonous increase in the shear viscosity.The increase in the shear viscosity of thesolution with an increase in the solutionconcentration was due to the increased

Chiang Mai J. Sci. 2011; 38(1) 61

likelihood for chain entanglements [30]. Anincrease in the solution concentration, on theother hand, resulted in a monotonous decreasein the electrical conductivity. As pointed outby a number of researchers [1,3], the electricalforces that influence the ejection and thestretching of the ejected, charged jet aregoverned by the amount of unassociatedcharges within a jet segment. These unassociatedcharges, believed to be carried over by water,are in the form of residual ionic impurities[1,3], provided that none of the majorsubstances in the spinning solution did notundergo a dissociation reaction into ionicspecies upon subjection to a high electricalpotential. An increase in the solutionconcentration caused the amount of thesolvent (i.e., 80% v/v ethanol aqueoussolution), hence that of water, to decrease.The gradual decrease in the amount of waterresulted in a hypothetic decrease in the amountof the unassociated charges, hence theobserved reduction in the electricalconductivity. Notwithstanding, since theamount of water in the ethanol aqueoussolution was only 20% v/v, the decrease inthe electrical conductivity of the solutions wasnot significant.

To obtain the condition that resulted inthe electrospun zein fiber mats with optimizeddiameters, the zein solutions with concentra-tions in the range of 30 to 45% w/v wereelectrospun under applied potentials in therange of 15 to 25 kV and collection distances

in the range of 10 to 20 cm for 5 min.Table 2 shows representative SEM images ofthe electrospun fiber mats from the zeinsolutions that had been fabricated under theapplied potential and the collection distanceof 20 kV and 15 cm, respectively. Notwith-standing, the SEM images of the electrospunfiber mats from the zein solutions that hadbeen fabricated under all of the investigatedconditions are provided as Supplementaryinformation. According to the obtainedresults, the diameters of the electrospun zeinfibers that had been obtained under theseconditions ranged between 0.32 and 1.10 μm.For the zein solutions with the concentrationslower than 35% w/v, beaded fibers were thecommon features. For given values of theapplied electrical potential and collectiondistance, increasing concentration of the zeinsolutions caused the diameters of the fibersto increase. For the beaded fibers, an increasein the concentration of the zein solutionsresulted in decreased tendency for beadformation. For given values of the solutionconcentration and the collection distance, anincrease in the applied electrical potentialresulted in an increase in the fiber diameters.For given values of the solution concentrationand the applied electrical potential, an increasein the collection distance generally resulted ina reduction in the fiber diameters, except forthe fibers that had been obtained from the45% w/v zein solution.

The results were in general accordance

Solution concentration Shear viscosity Electrical conductivity(wt.%) (mPa s) (μμμμμS cm-1)

30 120 ± 1 884 ± 135 188 ± 1 859 ± 140 289 ± 2 850 ± 145 491 ± 1 815 ± 1

Table 1. Shear viscosity and electrical conductivity of the as-prepared zein solutions at 25oC(n = 3).

62 Chiang Mai J. Sci. 2011; 38(1)

with that previously reported by Torres-Gineret al. [27] Increasing the solution concentrationto a value that is beyond a critical, chain-overlapping concentration results in extensivechain entanglements that are enough to preventthe total break-up of the ejected, charged jetinto discrete drops [30]. Further increase inthe solution concentration results in themonotonous increase in the chain entangle-ments that contribute directly to the observedincrease in the viscosity of the solutions, hencethe increase in the viscoelastic forces thatcounteract the electrical forces responsible forthe stretching or the thinning of the chargedjet segments. In addition, increasing theviscosity of the solutions causes the onset pointof the bending instability to occur closertowards the collector, resulting in thereduction of the total path lengths for whichthe jet segments have to travel to the collector.An increase in the electrical potential also causesthe onset point of the bending instability tooccur closer towards the collector, which is adirect result of the increase in the electrostaticforces. On the other hand, an increase inthe collection distance results in a reduction inthe electrostatic forces exerting on the jetsegments, causing the bending instability to

occur closer to the nozzle tip, which, in turn,increases the total parth lengths for whichthe jet segments have to travel to the collector[30].

Since the electrospun zein fiber mats cannot sustain their fibrous structure when beingsubmerged in distilled water as well as inethanol, cross-linking of these fiber mats isnecessary. According to Table 2, the electro-spinning condition that was chosen to obtainfiber mats for further investigations was 40%w/v zein solution that was electrospun underthe fixed electric field of 20 kV/15 cm. Thiscondition was selected because it was easy tospin and no discrete droplets of zein wereobserved during the electrospinning. Thecross-linked electrospun zein fiber mats wereprepared by adding varying amounts of theglyoxal aqueous solution (i.e., 0, 5, 10 and 15%v/v based on the volume of the zein solution)to the 40% w/v zein solution. Previously, Yaoet al. [26] reported successful cross-linkingof electrospun zein fiber mats with HDI,by immersing the electrospun fiber matsin tetrahydrofuran (THF) containing 1 wt.%HDI for 10 h. Selling et al. [28] usedglutaraldehyde as the cross-linking agent andthe curing of the glutaraldehyde-containing

Table 2. Representative SEM images (scale bar = 5 μm; collection time = 5 min) illustratingthe effect of concentration of the zein solutions on morphology of the obtained fibers at afixed electric field of 20 kV/15 cm that had been collected for 5 min as well as average valuesof diameters, bead sizes, and number of beads per unit area (i.e., bead density) of the obtainedfibers.

Solution concentration (% w/v)

30 35 40 45

Fiber diameters (μm) 0.46 0.11 0.60 0.12 0.74 0.13 1.05 0.27Bead sizes (μm) 1.59 0.55 - - -Bead density (10-4 μm-2) 7.61 2.53 - - -

Chiang Mai J. Sci. 2011; 38(1) 63

zein fiber mats was done at differenttemperatures from 80 to 180oC for varioustime intervals in order to obtain the cross-linked fiber mats with various degrees ofinsolubility. Here, the approach of Selling etal. [28] was followed, but glyoxal was usedinstead of glutaraldehyde. Prior to electro-spinning, the glyoxal-containing zein solutionswere characterized for their shear viscosity andelectrical conductivity and the results are shown

in Table 3. Clearly, the addition of glyoxal inthe zein solution caused the shear viscosity todecrease and, with increasing the glyoxalcontent in the solutions, the shear viscositydecreased slightly. On the other hand, theelectrical conductivity decreased markedly withthe initial addition of glyoxal in the solution,but the property values did not changedsignificantly with further increase in the glyoxalcontent in the solutions.

Type of zein solution Shear viscosity Electrical conductivity(mPa s) (μμμμμS cm-1)

Neat 284 ± 2 850 ± 1With 5% v/v glyoxal 266 ± 2 929 ± 3With 10% v/v glyoxal 253 ± 1 929 ± 1With 15% v/v glyoxal 252 ± 1 943 ± 1

Table 3. Shear viscosity and electrical conductivity of the as-prepared zein solutions (40 wt.%)with and without the presence of 0.1 M of glyoxal aqueous solution in various amounts at25oC (n = 3).

Certain mechanical properties, e.g.,Young’s modulus, tensile strength, andpercentage of elongation at break, of the neatand the cross-linked electrospun zein fibermats were investigated and the results aresummarized in Table 4. The results indicatedthe dramatic improvement in the mechanicalperformance of the cross-linked fiber matsover that of the neat materials. Among thecross-linked materials, the initial increase in theglyoxal content used to cross-link the zein fibermats (up to 10% v/v) resulted in the increasein all of the values of the tested mechanicalproperties. Further increase in the glyoxalcontent to 15% v/v, the property valuesdropped quite dramatically. This could be aresult from the dilution of the zein solution inresponse to the addition of a larger quantityof the glyoxal aqueous solution. Clearly, the

zein fiber mats that had been cross-linked atthe glyoxal content of 10% v/v exhibited thegreatest property values (i.e., Young’s modulus= 3,560 MPa, tensile strength = 2.2 MPa, andelongation at break = 6.7%, on average).Yao et al. [26] showed that, among the variousHDI-cross-linked zein fiber mats, the onesthat obtained from the 40% w/v zein solutionin 70:30 v/v ethanol/water showed thegreatest Young’s modulus and tensile strengthvalues of 95.2 and 4.2 MPa, respectively.Clearly, the cross-linked zein membranes asobtained in this work exhibited much greaterstiffness, despite the lower tensile strengthvalue. It should be noted, however, thatmeasurements that relate to the mechanicalproperties of electrospun fibrous membranesare often hampered by the wide variationsof the obtained data.

64 Chiang Mai J. Sci. 2011; 38(1)

The stability of both the neat and thecross-linked electrospun zein fiber mats upontheir submersion in an aqueous medium needsto be determined if they are to use in certainapplications that requires a contact with suchmedium. Some of these applications are, forexamples, scaffolds for cell/tissue culture.Table 5 shows morphology of the neat andthe cross-linked electrospun zein fiber matsafter their submersion in distilled water atroom temperature (i.e., 25 ± 1oC) and 37oCfor 1 and 5 d. Apparently, the neat zein fibermats practically lost their fibrous nature whenthey were submerged in distilled water at alltemperatures and time points investigated,while all of the cross-linked zein fiber matsappeared to lose their fibrous nature whenthey were submerged in distilled water at 37oCfor 5 d. Despite the improved stability indistilled water, the physical character of thecross-linked fibers changed from ribbon-liketo become flattened and even fused toadjacent fibers at touching points. Evidently,the flattening and the fusing of the fibersbecame more obtained for the case of theneat zein fibers, as the physical character ofthe membranes resembled that of the solvent-cast films with embedded porous structure.To further illustrate the effect of thesubmersion time interval (i.e., 1, 2, 3, 4, or5 d) on morphology of the neat and the cross-

linked electrospun zein fibrous membranes,additional representative SEM images aresupplied as Supplementary information.

To assess whether the neat and the cross-linked electrospun zein fiber mats could beused as scaffolding materials for cell/tissueculture, indirect cytotoxicity evaluation wascarried out on these materials. Figure 1a showsthe viability of mouse fibroblasts (L929) thatwere cultured with the extraction media thathad been prepared from the neat and thecross-linked electrospun zein fiber mats for3 d against that of the cells that were culturedwith the fresh SFM. Two groups of thesamples were investigated: without and withthe treatment with glycine. Without the glycinetreatment, the neat electrospun zein fiber matsreleased no substance in the level that was toxicto the cells, as the viability of the cultured cellswas similar to that of the control. However,as the glyoxal content used to cross-link thematerials increased, the viability of the cellsdecreased from about 95% (for the fiber matsthat had been cross-linked with 5% v/vglyoxal) to about 42% (for the fiber mats thathad been cross-linked with 15% v/v glyoxal).Upon treating the samples with glycine, theviabilities of the cultured cells were significantlygreater and, even for the fiber mats that hadbeen cross-linked with 15% v/v glyoxal, theviability of the cells appeared to be the same

Table 4. Mechanical properties in term of Young’s modulus, tensile strength and percentageof elongation at break of the neat and the cross-linked electrospun zein fiber mats (i.e., ZP,ZP5, ZP10 and ZP15 denote the fiber mats that had been obtained from the zein solutionscontaining 0, 5, 10 and 15% v/v of the glyoxal aqueous solution, respectively) (n = 5).

Type of the electrospun Young’s modulus Tensile strength Elongation at breakzein fiber mats (MPa) (MPa) (%)

ZP 2,520 ± 300 0.9 ± 0.1 1.5 ± 0.4ZP5 3,080 ± 1200 1.5 ± 0.1 5.5 ± 1.6ZP10 3,560 ± 1600 2.2 ± 0.2 6.7 ± 2.0ZP15 2,980 ± 500 1.3 ± 0.3 1.4 ± 0.8

Chiang Mai J. Sci. 2011; 38(1) 65

Temperature Time Materials

( oC) (day) ZP ZP5 ZP10 ZP15

0

1

25

5

1

37

5

Table 5. The morphology of the neat and the cross-linked electrospun zein fiber mats aftersubmersion in distilled water at room temperature (i.e., 25 ± 1oC) and 37oC for 1 and 5 d.

as that of the control. Based on the obtainedresults, the effect of glycine may be two-fold.Firstly, the amino group of glycine helps toblock the unreacted aldehyde group of glyoxaland, secondly, the unreacted glycine acts asadditional supplement to the cells.

Figure 1b shows the viability of L929that were cultured with the extraction mediathat had been prepared from the neat and thecross-linked electrospun zein fiber mats for7 d against that of the cells that were culturedwith the fresh SFM. The two groups of the

samples were also investigated here. Withoutthe glycine treatment, the neat electrospun zeinfiber mats still released no substance in thelevel that was toxic to the tested cells. Similarly,as the glyoxal content used to cross-link thefibrous membranes increased, the viability ofthe cells decreased from about 93% (for thefiber mats that had been cross-linked with 5and 10% v/v glyoxal) to about 81% (for thefiber mats that had been cross-linked with15% v/v glyoxal). Compared with the viabilityof the cells that were cultured with the

66 Chiang Mai J. Sci. 2011; 38(1)

extraction medium that had been preparedfrom the fiber mats that were cross-linkedwith 15% v/v glyoxal for 3 d, that of thecells that were cultured with the extractionmedium that had been prepared from the samematerials for 7 d was unexpectedly greater.This could be a result of the change in the

chemical structure of the toxic species thatwas released into the medium to the non-toxicone during prolonged incubation. For theglycine-treated samples, the viabilities of thecultured cells were considerably greater thanthose of the cells that had been cultured withthe fresh medium and the extraction media

Figure 1. Viability of mouse fibroblasts (L929) that were cultured with extraction mediaprepared from the neat and the cross-linked electrospun zein fiber mats that had been submergedin fresh serum-free medium (SFM) for (a) 3 and (b) 5 d against that of the cells that werecultured with the fresh SFM (n = 3).

(a)

(b)

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from the non-treated samples. This shouldbe a result of the much greater amounts ofglycine that had been released into theextraction media during the prolongedincubation, as the greater amounts of glycinehelped to promote the growth of the cells[31], in addition to the blocking of theunreacted aldehyde group of glyoxal [32].

The potential for use of the cross-linked

electrospun zein fiber mats as scaffolds forcell/tissue culture was evaluated with the fibermats that had been cross-linked with 10%v/v glyoxal. Figure 2 shows representativeSEM images at two magnifications of humanforeskin fibroblast cells (HFF) that had beencultured on the substrates for 5 d. In the lowmagnification image (350x), it can be seen thata good number of cells attached well on the

Figure 2. Representative SEM images of human foreskin fibroblast cells (HFF) that had beencultured on the cross-linked electrospun zein fiber mats (i.e., ZP10) for 5 d at two magnificationsof (a) 350x and (b) 2,000x.

(b)

(a)

68 Chiang Mai J. Sci. 2011; 38(1)

surface of the cross-linked fibrous membranes.It is also seen that the cells, after 5 d of cellculturing, reached the confluence to form thehypothetical monolayer construct on thefibrous membrane surface. Interestingly, in theempty, uncovered space of the substratesurface, the fibrous structure was clearly seenand this confirmed the legitimacy of thecross-linking procedure utilized here. In thehigh magnification image (2,000x), theexpansion of the cytoplasm of the attachedcells was clearly observed. Based on theobtained results, it is confirmed that the chosencross-linked electrospun zein fiber mats werenon-toxic to the cells and could be used assubstrates for cell/tissue culture.

4. CONCLUSIONUltra-fine zein fibers were successfully

prepared by electrospinning from zeinsolutions in 80% v/v ethanol aqueous solution.Various processing conditions (i.e., solutionconcentration, applied potential and collectiondistance) were investigated to obtain theoptimal condition for electrospinning. Beadedfibers were the common features for the zeinsolutions with concentrations lower than 35%w/v. The diameters of the individual fibersegments were found to increase with anincrease in either the solution concentrationor the applied potential, while they decreasedwith an increase in the collection distance. Anincrease in the concentration of the zeinsolutions also resulted in less tendency for beadformation. In all of the spinning conditionsinvestigated, the diameters of the obtainedfibers ranged between 0.32 and 1.10 μm.Cross-linking of the electrospun zein fibermats, in an attempt to preserve their fibrousstructure upon submersion in an aqueousmedium, was carried out by adding varyingamounts of glyoxal aqueous solution (i.e., 0,5, 10, and 15% v/v, based on the volume ofthe zein solution) to 40% w/v zein solution.

Cross-linking improved the mechanicalintegrity of the materials and increasing theamount of glyoxal aqueous solution in thebase zein solution resulted in observedincreases in the mechanical properties,excepted for the cross-linked fiber mats fromthe solution that contained 15% v/v glyoxalaqueous solution that showed equivalentvalues to those of the neat materials. Theimprovement in the physical appearance ofthe cross-linked electrospun zein fiber matswas evident upon their submersion in distilledwater at either room temperature (i.e., 25 ±1oC) or 37oC for 1 or 5 d, as the underlyingfibrous structure was still intact, despite theobservation of the fusing and the flatteningof the fibers. The potential for use of thecross-linked electrospun zein fiber mats asscaffolding materials for cell/tissue culture wasassessed by the indirect cytotoxicity evaluationusing mouse fibroblasts (L929) and the resultsshowed that the toxicity level increased withan increase in the amount of glyoxal aqueoussolution that was used to cross-link thematerials. Notwithstanding, such the toxicitycould be alleviated with the treatment withglycine. Finally, the zein fiber mats that hadbeen cross-linked with 10% v/v glyoxalaqueous solution supported the growth ofhuman foreskin fibroblasts (HFF) relativelywell.

ACKNOWLEDGEMENTSThe authors acknowledged partial

support from the Center for Petroleum,Petrochemicals and Advanced Materials(C-PPAM) and the Petroleum and Petro-chemical College (PPC), ChulalongkornUniversity and the Thailand Research Fund(DBG5280015).

Chiang Mai J. Sci. 2011; 38(1) 69

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