676 Chiang Mai J. Sci. 2014; 41(3)

Chiang Mai J. Sci. 2014; 41(3) : 676-690http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Poly(3-hydroxybutyrate)/magnetite CompositeNanofibers Obtained Via Combined Electrospinningand Ammonia Gas-enhancing In SituCo-precipitation: Preparation and Potential Use inBiomedical ApplicationsPakakrong Sangsanoh [a,b] and Pitt Supaphol*[a,b][a] The Petroleum and Petrochemical College,Chulalongkorn University, Phyathai Road, Pathumwan,

Bangkok 10330, Thailand.[b] The Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University,

Phyathai Road, Pathumwan, Bangkok 10330, Thailand.*Author for correspondence; e-mail: pitt.s@chula.ac.th

Received: 17 April 2013Accepted: 11 July 2013

ABSTRACTIn the present study, we developed a relatively simple way to incorporate magnetic

nanoparticles into electrospun ultrafine fibers of poly(3-hydroxybutyrate) (PHB) by combiningelectrospinning with in situ co-precipitation. Using this approach, we achieved uniform dispersalof Fe3O4 nanoparticles along the fibrous surface. The formation of Fe3O4 nanoparticles onthe fiber surface and the particle size and distribution were investigated by scanning electronmicroscopy (SEM), X-ray diffraction (XRD) and energy dispersive X-ray (EDX). The contentof Fe3O4 nanoparticles in the composite nanofibers was analyzed by thermogravimetric analysis(TGA). Potential uses in biomedical applications were evaluated by an indirect cytotoxicity testusing L929 mouse fibroblasts and murine neuroblastoma Neuro2a cells.

Keywords: electrospinning, poly(3-hydroxybutyrate), in situ co-precipitation, magneticnanoparticle, indirect cytotoxicity

1. INTRODUCTIONRecent advances in nanotechnology have

greatly expedited the development ofmagnetically responsive hybrid materials thatexhibit magnetic field-dependent behaviorthat may have potential uses in a wide rangeof biomedical applications [1-6]. Magnetismcan be produced by incorporating magneticagents into a polymer matrix. Among thevarious types of magnetic agents, a black iron

oxide particle called magnetite (Fe3O4) exhibitsthe strongest magnetism of any transition metaloxide [7] and has been extensively studiedbecause of its biocompatibility and lowcytotoxicity in living cells [3, 8]. The size ofthe particles plays an important role in theirmagnetic properties [9], as particles offerromagnetic materials no longer exhibitthe cooperative bulk phenomenon of

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ferromagnetism when their diameter is lessthan 100 nm. Rather, such nanoparticlesexhibit superparamagnetism that no longerexhibits hysteresis [10]. According to thissuperparamagnetic behavior, such nanoparti-cles should also deform in the direction ofthe external magnetic field gradient andshould no longer show magnetism withoutan external magnetic field. These changes indirection should be completely reversiblebecause the magnetic moment of particlesshould relax to the original distributionafter removal of the magnetic field [2,10].

Various approaches have been utilizedto incorporate superparamagnetic Fe3O4

nanoparticles into nanofibers. Electrospinningis a versatile and effective method for theproduction of polymeric nanofibers withdiameters ranging from a few micrometersdown to several hundred nanometers. Theseelectrospun fibrous substrates exhibit severalinteresting characteristics, such as high ratio ofsurface area to mass or volume, a small inter-fibrous pore size with high porosity and vastpossibilities for surface functionalization;therefore, electrospun polymeric fibers aregood candidates for controlled drug deliveryand tissue engineering applications [11,12]. Inaddition, electrospinning has been demons-trated to be a simple way to prepare compositenanofibers through electrospinning of aFe3O4 nanoparticle-filled polymer solution.Several studies have demonstrated thepossibility of producing compositenanofibers by incorporating Fe3O4

nanoparticles into polymer solutions suchas poly(lactic acid) [13], poly(methylmethacrylate) [14, 15], polyaniline [16],poly(ethylene oxide) [10], polyacrylonitrile[17-20], poly(vinyl pyrrolidone) [21, 22], poly(vinylidene fluoride) [23], poly(acrylonitrile-co-acrylic acid) [24], poly(vinyl chloride) [25]and poly(vinyl alcohol) [26-29]. However, thistraditional strategy has some disadvantages,

such as stepwise processing and theagglomeration of particles because of thestrong Van der Waals force, which makesit difficult to re-disperse the particles wellin the spinning solution. The agglomerationcan only be minimized by coating theparticle surface with steric stabilizers oradsorbing surfactants [10, 29, 30].

In the present study, we developeda novel approach to prepare poly(3-hydroxybutyrate)(PHB)/magnetitecomposite nanofibers by combining theelectrospinning technique with an ammoniagas-enhancing in situ co-precipitationmethod. PHB is a biodegradable andbiocompatible thermoplastic polyesterproduced by various microorganisms thatcompletely degrades to release a normalcomponent of blood and tissue, d, l-b-hydroxybutyrate (HB) [31]. These outstandingproperties render PHB as a good candidatefor biomedical applications. The propertiesof an as-prepared solution and themorphology of the obtained PHB-Fe3O4

composite nanofibers were characterized.The existence of Fe3O4 nanoparticles andthe magnetic properties of PHB-Fe3O4

composite nanofibers were also investigated.The potential use of these composite fibermats as scaffolding materials was evaluatedin vitro using mouse fibroblasts L929 cell lineand murine neuroblastoma Neuro2a cell line,in which indirect cytotoxicity was analyzed.The PHB-Fe3O4 composite nanofibers werecompared with tissue-culture polystyrene(TCPS) plates and pristine as-spun PHB fibermats.

2. MATERIALS AND METHODS2.1 Materials

Materials used in the fabrication of thePHB-Fe3O4 composite nanofibers includedpoly(3-hydroxybutyrate) (PHB; MW =300,000 g×mol-1; Sigma-Aldrich, USA),

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poly(ethylene oxide) (PEO; MW = 600,000g×mol-1; Acros Organics, Thailand), iron (III)chloride hexahydrate (Honeywell Riedel-deHa n®, Germany), iron (II) chloridetetrahydrate (Honeywell Riedel-de Ha n®,Germany) and 30 wt% ammonia (Lab-scan,Asia). Chloroform (Lab-scan, Asia) anddistilled water were used as solvents. Distilledwater was deoxygenated by bubbling nitrogengas prior to use. All chemicals were useddirectly without further purification.

2.2 Preparation of Spinning Solutions andElectrospinning Experiments

The electrospun PHB-Fe3O4 compositenanofibers were fabricated by using theelectrospinning technique combined withan ammonia gas-enhancing in situ co-precipitation method, as summarized inSchematic 1. Briefly, 20% w/v PHB wasdissolved in chloroform and stirred for 4 hat 60°C. Additionally, 1% w/v aqueousdeoxygenated PEO solution containing ironions was prepared by dissolving variousamounts of iron ions (Fe3+ and Fe2+ with a

fixed ratio of 2:1) into a PEO solution andstirring for 6 h at 60°C to form the hybridsolutions. The total concentration of iron inthe aqueous iron ion solutions was varied tobe 0.05 M, 0.10 M, 0.20 M and 0.30 M. Whencompletely dissolved, the hybrid solutionswere mixed into the PHB solution with aweight ratio of 1:9 to obtain the as-preparedspinning solutions, and these solutions werestirred vigorously overnight at 60°C under anitrogen atmosphere. Each of the spinningdopes was subsequently filled into a 20-mlglass syringe, where the open end wasconnected to a 20 gauge stainless steel needle(OD= 0.91 mm) that was used as the nozzle.A rotating drum (width and OD of thedrum ≈ 15 cm; rotational speed = 200 rpm)was used as the collector. The outer surfaceof the rotating drum was covered with analuminum sheet and set approximately 20cm from the tip of the needle. A GammaHigh Voltage Research DES30PN/M692power supply was used to generate a fixedDC potential of 15 kV. The collection timewas also fixed at approximately 4 h.

Schematic 1. Summarizes the pathway for the preparation of PHB-Fe3O4 compositenanofibers by using the electrospinning technique in combination with an ammoniagas-enhanced in situ co-precipitation method.

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Fe3O4 nanoparticles were synthesizedonto the as-spun PHB nanofibers by usingthe ammonia gas-enhancing in situ co-precipitation method. Briefly, the electrospunPHB containing iron ions was kept insidethe reaction vessel and pre-treated withnitrogen gas for 10 min to eliminate oxygengas in the porous fibers. Then, the fiber matswere subsequently exposed to the ammoniaatmosphere for 10 min to start the formationof Fe3O4 nanoparticles, which caused thecolor of the fiber mats to gradually changefrom yellow to dark brown. The electrospunPHB-Fe3O4 composite nanofibers were rinsedsuccessively with distilled water until theywere neutral to remove the residual ammonia.Finally, the composite fiber mats were driedunder vacuum and kept in a desiccator untilfurther investigation.

2.3 CharacterizationPrior to electrospinning, each of the

spinning solutions was characterized withregard to viscosity and conductivity using aBrookfield DV-III programmable viscometerand an Orion 160 conductivity meter,respectively. The morphology of the as-spunfiber mats before and after ammonia gastreatment was observed by using a JEOLJSM-5200 scanning electron microscope(SEM) with built-in energy dispersive X-rayanalysis (EDX). The formation of Fe3O4

nanoparticles was verified by X-ray diffraction(XRD) (Rigaku). The obtained compositenanofibers were scanned from 2q = 10° to2q = 70°. The content of Fe3O4 nanoparticlesin the composites was determined by using athermogravimetric analyzer (Perkin Elmermodel TGA7) in the temperature range from50 to 700°C with a heating rate of 10°C/minunder a nitrogen atmosphere (50 ml/min).The magnetic properties of the electrospuncomposite nanofibers were evaluated byusing a vibrating sample magnetometer

(VSM; LakeShore Model 7404). Themagnetization versus magnetic field (M-Hcurves or hysteresis loops) was plotted as afunction of applied magnetic field (Oe) atroom temperature (298 K).

2.4 Biological Compatibility EvaluationTo evaluate the potential use of

the electrospun PHB-Fe3O4 compositenanofibers in biomedical applications, theirbiocompatibility, in terms of indirectcytotoxicity toward mouse connectivetissue fibroblast-like cells (L929) andmurine neuroblastoma Neuro2a cells(American Type Culture Collection: ATCC),was evaluated in vitro in comparison withthe corresponding pristine electrospun PHBfiber mat and tissue-culture polystyrene(TCPS). Mouse connective tissue L929 cellswere cultured as a monolayer in Dulbecco’smodified Eagle medium (DMEM; Sigma-Aldrich) supplemented by 5% fetal bovineserum (FBS; Biochrom AG, Germany), 1%L-glutamine (Invitrogen Corp.), and 1%antibiotic and antimycotic formulation[containing penicillin G sodium, streptomycinsulfate, and amphotericin B (InvitrogenCorp.)]. For the murine neuroblastomaNeuro2a cell line, the cells were cultured asa monolayer in MEM/EBSS medium(HyClone), supplemented by 10% fetalbovine serum (HyClone), 2 mM L-glutamine(Gibco) and 1X Pen/Strep (Gibco). The cellswere incubated at 37°C in a humidifiedatmosphere containing 5% CO2, and theculture medium was replaced once every2 d. The reference cells from the cultureswere trypsinized [0.25% Trypsin-EDTA(Gibco)] and seeded on TCPS.

The indirect cytotoxicity of electrospunPHB-Fe3O4 composite nanofibers wasevaluated in an adaptation of the ISO10993-5 standard test method. First, the extractionmedium was prepared by immersing

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specimens cut from the composite fiber mats(~7 mm in diameter) in wells of a 96-welltissue culture plate in a serum-free medium(SFM; containing DMEM, 1% L-glutamine,1% lactalbumin, and 1% antibiotic andantimycotic formulation) and incubatingthem for 1 d, 3 d and 5 d. To prepare thereference cells, L929 and Neuro2a cells wereseeded in the wells of a 96-well tissue cultureplate at a density of 1.0 × 104 cells/well andincubated in 5% SFM to allow cell attachmenton the plate. After 24 h, the culture mediumwas removed and the as-prepared extractionmedium was added to the wells. The cellswere further incubated for 24 h, afterwhich the amount viable cells was measuredusing a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.The viability of cells that were cultured withfresh SFM was used as a control.

2.5 Quantification of Viable Cells byMTT Assay

The MTT assay is used to quantify theamount of viable cells based on the reductionof yellow tetrazolium salt to purple formazancrystals by dehydrogenase enzymes secretedfrom the mitochondria of metabolically activecells. The amount of purple formazan crystalsformed is proportional to the number ofviable cells. First, the culture medium of eachcultured specimen was removed andreplaced with 100 μL/well of MTT solution(Sigma-Aldrich) at 5 mg×mL-1 for a 96-welltissue culture plate (or 500 μL/well for a 24-well tissue culture plate), and then the platewas incubated for 1 h. After incubation,the MTT solution was removed. Then,100 μL/well of dimethyl sulfoxide (DMSO;Riedel-de Ha n, Germany) was added todissolve the formazan crystals (or 500 μL/well for a 24-well TCPS), and the plate wasleft at room temperature in the dark for 1 hon a rotary shaker. Finally, the absorbance

at 570 nm, representing the proportionof viable cells, was recorded usinga Thermo Labsystems (Multiscan Ex)spectrophotometer.

3. RESULTS AND DISCUSSIONComposite nanofibers containing Fe3O4

nanoparticles embedded into a polymerare of particular interest in the fieldof biotechnology and medicine becauseof their magnetic field-dependent physicalproperties. Among fiber fabricationtechniques, electrospinning is a versatile andeffective method to prepare fibers withdiameters ranging from micrometersdown to a few nanometers, and it provides asimple way to incorporate the nanoparticlesinto the polymer matrix. However, themajor difficulty in the preparation of suchcomposite nanofibers is the formation of astable dispersion of nanoparticles in thecontinuous polymer matrix. Here, we reporta novel approach where Fe3O4 nanoparticleswere embedded into composite nanofibersby combining electrospinning with ammoniagas-enhanced in situ co-precipitation.

3.1 Preparation and Characterization ofElectrospun Fibers Containing Iron Ionsand the Obtained Electrospun PHB-Fe3O4 Composite Nanofibers

It is common knowledge that theproperties of the spinning solutions play animportant role in the morphologicalappearance of the obtained electrospunnanofibers. In the present study, the presenceof iron ions in the as-prepared solutionssignificantly changed the conductivity andviscosity, as shown in Figure 1. For a givenpolymer concentration, the viscosity ofsolution decreased and the conductivityincreased with the addition and increasingconcentration of iron ions. Therefore, theadded iron ions had a clear effect on the

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spinning solution. Table 1 shows selectedSEM images of electrospun pristine PHBand electrospun PHB containing iron ionsthat were produced under a constantelectrical potential and over a fixed collectiondistance. Evidently, the addition of iron ionsresulted in the formation of smaller fibers.The fiber diameters decreased graduallyfrom 2.68 ± 0.79 mm with pristine PHB to2.16 ± 0.68 mm and 1.31 ± 0.53 mm withincreasing iron ion concentrations upto approximately 0.05 M and 0.10 M,respectively. When the concentration ofaqueous iron ions was increased to 0.20 M,the fiber diameter decreased to 0.68 ± 0.29mm with bead formation. Preliminary studiesshowed that at concentrations of aqueousiron ions higher than 0.30 M, electrospinningresulted only in the formation of discretebeads (data not shown). The increase insolution conductivity and decrease insolution viscosity contributed to the observeddecrease in fiber diameter and ability toform continuous fibers because of theCoulombic repulsion of charges presentwithin the jet segment and the lowermechanical resistance when the fibers

experience bending instability [31]. In thecase of bead formation, the degree of chainentanglement is not high enough towithstand the Coulombic stretching forceacting on the jet segment, which causes thejet to break into smaller jets that are laterrounded up to form beads [32]. For furtherstudy, the as-prepared solution of pristinePHB and the PHB solution containing ironions were electrospun continuously for 4 h toproduce fiber mats with thicknesses ofapproximately 192 ± 25 mm. The as-spunfibers containing iron ions had a yellow surfacebecause of the incorporation of iron ions.After ammonia gas treatment, the color ofthe as-spun fibers containing iron ions changedfrom yellow to dark brown, which indicatedthe formation of Fe3O4 nanoparticles in thecomposite nanofibers. A possible formationmechanism of magnetite particles is asfollowing (see Schematic 2): (1) at thebeginning, the reactant mixtures of Fe2+ andFe3+ existed on the fibrous surface; (2) withexposing OH- of ammonia solution, theformation of Fe(OH)3 and Fe(OH)2 wasoccurred; (3) the Fe(OH)3 and Fe(OH)2

dehydrated and formed Fe3O4 crystals [33].

Figure 1. Viscosity and conductivity of the as-prepared PHB solution as a function of aqueousiron ion concentration. The as-prepared solution prepared from 20% w/v PHB in chloroformmixed with 1% w/v aqueous deoxygenated PEO solution contained various amounts of ironions (Fe3+ and Fe2+ with a fixed ratio at 2:1) with a weight ratio of 1:9.

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Table 1. Selected SEM images of the as-spun pristine PHB, PHB containing iron ions andcorresponding PHB-Fe3O4 composite nanofibers that were produced under a constant electricalpotential of 15 kV and over a fixed collection distance of approximately 20 cm.

Schematic 2. A possible formation mechanism of magnetite particles.

Aqueous iron ion

concentration (M)

Pristine PHB

0.05 M iron ions

0.10 M iron ions

0.20 M iron ions

Scale bar = 10 μm

Magnification = 1000×

Scale bar = 1 μm

Magnification = 10000×

After ammonia treatment

Scale bar = 1 μm

Magnification = 10000×

Before ammonia treatment

Fiber diameter: 2.68 ± 0.79 μm

Fiber diameter: 2.16 ± 0.68 μm Particle size: 67.62 ± 15.40 μm

Fiber diameter: 1.31 ± 0.53 μm Particle size: 75.33 ± 21.39 μm

Fiber diameter: 0.68 ± 0.29 μm Particle size: 82.81 ± 25.74 μm

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Table 1 shows selected SEM images ofthe electrospun PHB-Fe3O4 compositenanofibers prepared using solutions containingaqueous iron ions at concentrations of0.05 M, 0.10 M and 0.20 M. The SEMimages show the regular shapes of the Fe3O4

nanoparticles distributed uniformly alongthe fibrous surface. The distribution of Fe3O4

nanoparticles on the fiber surface may be a

result of the local electric field generated bythe electrospinning process [10]. The averageparticle sizes of Fe3O4 nanoparticles thatwere distributed on the fiber surfaces wereapproximately 67.62 ± 15.40 nm, 75.33 ±21.39 nm and 82.81 ± 25.74 nm for iron ionconcentrations of 0.05 M, 0.10 M and 0.20M, respectively. The increase in the averageparticle size with increasing concentrations of

The formation of Fe3O4 nanoparticlesin the PHB-Fe3O4 composite nanofibers wasconfirmed by X-ray diffraction (XRD)analysis, and the diffraction patterns arepresented in Figure 2. From the literature[24], the characteristic diffraction patternsof pure Fe3O4 crystals are observed at2q = 30.40°, 35.81°, 43.53°, 54.02°, 57.59°and 63.25°, corresponding to the (2 2 0),(3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0)planes, respectively. For the electrospunpristine PHB fiber mat, the characteristicdiffraction pattern peaks were observed at2q = 12.4°, 16°, 20° and 23°, correspondingto the diffraction patterns of the crystallinePHB polymer powder [34]. All diffraction

patterns of the obtained electrospunPHB-Fe3O4 composite nanofibers were inagreement with the characteristic XRDpatterns of pure Fe3O4 nanoparticles,which indicate that Fe3O4 nanoparticles hadformed after ammonia gas treatment.However, the peak intensity of the Fe3O4

nanoparticles decreased and the full width ofthe peak increased, which indicates lowcrystallinity and small crystal size. These resultsmay be predominantly attributed to theexistence of the polymer matrix, whichdecreased the ability to form Fe3O4

nanoparticles and thereby caused a decreasein the crystal size and crystallinity of thenanoparticles [28].

Figure 2. Wide-angle X-ray diffraction patterns of Fe3O4 nanoparticles (a), pristine PHB (b),PHB-Fe3O4 composite nanofibers prepared using aqueous iron ion solution with an ironconcentration of 0.05 M (c), 0.10 M (d) and 0.20 M (e).

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iron ions has also been reported by Deepaet al. [35]. The existence of Fe3O4 nanoparticlesthroughout the fibers was also confirmedby energy dispersive x-ray (EDX) analysis.Figure 3 shows the EDX analysis of thecomposite nanofibers using iron ions at a

concentration of 0.10 M. Several strongpeaks at 0.7 eV, 6.4 eV and 7.1 eV,representative of Fe, were observed;therefore, Fe3O4 nanoparticles also exist inthe surface layer of the electrospuncomposite nanofibers.

Figure 3. The EDX pattern of Fe3O4 nanoparticles present on the surface of compositenanofibers prepared using 0.10 M iron ion solution.

The content of Fe3O4 nanoparticlesincorporated into the composite nanofiberswas further examined by thermogravimetricanalysis (TGA). To minimize the possibilityof weight increase as a result of iron oxidationand to allow the thermal decomposition ofthe polymer, these analyses were performedunder nitrogen atmosphere. The pristinePHB fiber mats exhibited only one-stagedecomposition from 200°C to 280°Cbecause of the breakdown of the polymerbackbone without any residues. For thefibrous composites, the TGA thermogramexhibited a two-stage decomposition,of which the first stage, observed up to~120°C, was caused by the removal ofadsorbed moisture, which corresponded toa weight loss of ca. 3%. The second stepof decomposition observed up to ~230°Ccorresponding to the degradation of thepolymer matrix was also observed.The remaining weight of the residues waspresent as Fe3O4 nanoparticles. The theoreticalamount of Fe3O4 nanoparticles that could becreated by a given amount of iron ion

reactants was calculated to be 16.46 wt%,27.64 wt% and 36.53 wt% for iron ionconcentrations of 0.05 M, 0.10 M and0.20 M, respectively. However, the actualamount of Fe3O4 nanoparticles in the fibrouscomposite that was estimated from theresidual mass percentages was approximately2.5 wt%, 6 wt% and 11 wt%, respectively, asshown in Figure 4. The significant differencein the theoretical and actual amounts of Fe3O4

nanoparticles may have been caused byinhibition of the growth of the nanoparticlesby the polymer matrix.

The superparamagnetic behavior ofelectrospun PHB-Fe3O4 composite nanofibersat room temperature is especially useful forapplications in nonuniform magnetic fieldsbecause of the ability to significantly reducedissipative energy. The magnetic hysteresiscurves of the composite nanofibers showsuperparamagnetic behavior with symmetrichysteresis and saturation magnetization atroom temperature, as is evident in Figure 5.As the magnetic field decreased, themagnetization decreased and reached zero

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with low remaining magnetization. Thesaturation magnetization (Ms) of thecomposite nanofibers was 0.13 emu�g-1, 0.44emu⋅g-1 and 0.60 emu⋅g-1 for iron ionconcentrations of 0.05 M, 0.10 M and 0.20M, respectively. However, the magnetizationof the bulk Fe3O4 reported in the literaturewas 90 emu⋅g-1[36]. The decrease inmagnetization in the electrospun PHB-Fe3O4 composite nanofibers is attributedto the following reasons: (1) the existenceof polymer matrices that encapsulate Fe3O4

nanoparticles and affect the effectivemagnetization of the nanoparticles [28]; and

(2) small particle size that results in thereduction of the magnetic moment in suchnanoparticles [9]. To obtain superparamagneticproperties, the remnant magnetization (Mr)and coercive field (Hc) of the electrospunPHB-Fe3O4 composite nanofibers shouldbe as low as possible. The remnantmagnetization of composite nanofibers atroom temperature was found to be 0.025emu⋅g-1, 0.105 emu⋅g-1, and 0.125 emu⋅g-1,whereas the coercive field was found to be12.53 G, 15.31 G and 17.87 G for iron ionconcentrations of 0.05 M, 0.10 M and0.20 M, respectively.

Figure 4. TGA thermograms of pristine PHB (a) and PHB-Fe3O4 composite nanofibersprepared using 0.05 M (b), 0.10 M (c) and 0.20 M (d) iron ion solutions.

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Figure 5. Magnetization versus applied magnetic field and a magnified view of the hysteresisloop at room temperature for PHB-Fe3O4 composite nanofibers prepared using 0.05 M (a),0.10 M (b) and 0.20 M (c) iron ion solutions.

3.2 Biological Compatibility EvaluationTo evaluate the potential use of

electrospun PHB-Fe3O4 compositenanofibers in biomedical applications, theirbiocompatibility in terms of indirectcytotoxicity toward L929 mouse fibroblastsand murine neuroblastoma Neuro2a cellswas evaluated in vitro in comparison with thecorresponding electrospun pristine PHBand tissue-culture polystyrene (TCPS).Cytotoxicity is a basic property of scaffoldingmaterials. Figure 6 shows the viability of thecells determined using an MTT assay afterthe cells had been cultured with extractionmedia obtained from electrospun pristinePHB fiber mats and PHB-Fe3O4 compositenanofibers mats, compared with viability of

cells cultured with fresh SFM. The viabilityof the cells was reported as a percentagerelative to the control. The viability of L929and Neuro2a cells cultured with the extractionmedia from all of the fibrous scaffolds wasequivalent to that of cells cultured with freshSFM, which indicates that the compositenanofibers do not release cytotoxic substancesto the culture media and implies that thesematerials are biocompatible toward L929 andNeuro2a cells. Previous reports repeatedlyshowed that Fe3O4 nanoparticles had goodbiodegradability and lacked cytotoxicity invitro and in vivo [1-4]. The present studyconfirmed that PHB-Fe3O4 compositenanofibers are promising candidates forbiomedical applications.

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Figure 6. Indirect cytotoxicity evaluation of PHB-Fe3O4 composite nanofibers in (a) mousefibroblasts (L929) and (b) murine neuroblastoma (Neuro2a) that were cultured with theextraction media from the nanofiber materials for 1 d, 3 d and 5 d. The viability of cells thatwere cultured with fresh culture medium (SFM) (i.e., control) was used as the reference toarrive at the viability of the attached cells shown in the figure.

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4. CONCLUSIONSIn this study, we successfully prepared

PHB-Fe3O4 composite nanofibers bycombining electrospinning technology withan in situ co-precipitation method. The Fe3O4

nanoparticles were synthesized in situ andwere well dispersed on the fibrous surface.The average particle size of the Fe3O4

nanoparticles was in the range of 67-82 nm.The particle size and particle size distributionof the Fe3O4 nanoparticles were controllableby adjusting the concentration of the aqueousiron ion solution. The obtained compositenanofibers had superparamagnetic propertieswith saturation magnetization values rangingfrom 0.13 to 0.60 emu⋅g-1 and very lowremnant magnetization (0.025-0.125 emu⋅g-1)and coercive fields (12-17 G) at roomtemperature. The potential use of theobtained composite nanofibers as scaffoldingmaterials for skin and nerve regenerationwas further assessed in L929 and Neuro2acells in terms of cytotoxicity by culturing thecells in media containing nanofiber extractsfor different immersion times. The viabilityof L929 and Neuro2a cells cultured with theextraction media from the compositenanofiber mats was equivalent to that of cellscultured with fresh SFM, which indicates thatPHB-Fe3O4 composite nanofibers havepotential for biomedical applications.

ACKNOWLEDGEMENTSThis work was supported in part by (1)

the Ratchadaphisek Somphot EndowmentFund for Research and Research Unit,Chulalongkorn University, and (2) the Centerof Excellence on Petrochemical and MaterialsTechnology (PETROMAT). (3) The NationalNanotechnology Center (NANOTEC)research fund (RES_54_198_63_006)P. Sangsanoh gratefully acknowledges thedoctoral scholarship (PHD/0191/2550)received from the Royal Golden Jubilee

Ph.D. Program, Thailand Research Fund(TRF).

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