http://informahealthcare.com/phdISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, Early Online: 1–8! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.954726

RESEARCH ARTICLE

Lysozyme-immobilized electrospun PAMA/PVA and PSSA-MA/PVAion-exchange nanofiber for wound healing

Prasopchai Tonglairoum, Tanasait Ngawhirunpat, Theerasak Rojanarata, and Praneet Opanasopit

Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand

Abstract

This research was aimed to develop the lysozyme immobilized ion-exchange nanofiber mats forwound healing. To promote the healing process, the PSSA-MA/PVA and PAMA ion-exchangenanofiber mats were fabricated to mimic the extracellular matrix structure using electrospin-ning process followed by thermally crosslinked. Lysozyme was immobilized on the ion-exchanenanofibers by an adsorption method. The ion-exchange nanofibers were investigated usingSEM, FTIR and XRPD. Moreover, the lysozyme-immobilized ion-exchange nanofibers werefurther investigated for lysozyme content and activity, lysozyme release and wound healingactivity. The fiber diameters of the mats were in the nanometer range. Lysozyme was graduallyabsorbed into the PSSA-MA/PVA nanofiber with higher extend than that is absorbed on thePAMA/PVA nanofiber and exhibited higher activity than lysozyme-immobilized PAMA/PVAnanofiber. The total contents of lysozyme on the PSSA-MA/PVA and PAMA/PVA nanofiber were648 and 166 mg/g, respectively. FTIR and lysozyme activity results confirmed the presence oflysozyme on the nanofiber mats. The lysozyme was released from the PSSA-MA/PVA andPAMA/PVA nanofiber in the same manner. The lysozyme-immobilized PSSA-MA/PVA nanofibermats and lysozyme-immobilized PAMA/PVA nanofiber mats exhibited significantly fasterhealing rate than gauze and similar to the commercial antibacterial gauze dressing. Theseresults suggest that these nanofiber mats could provide the promising candidate for woundhealing application.

Keywords

Immobilize, ion-exchange, lysozyme,nanofiber mats, wound healing

History

Received 3 July 2014Accepted 10 August 2014Published online 27 August 2014

Introduction

Wound recuperation has been important since the existence ofmankind. Disclosing wound may be exposed to microorganismand cause infection. Thus, the surface of the wound should not beabandoned to open and should be dressed with proper materials1.Moreover, these materials should be concordant with skin,oxygen-permeable, keep moist balance, protect the woundsagainst microorganisms and prevent them to reproduce1,2.

Due to an increasing interest in nanotechnology, nanofibershave gained much attention owing to their high specificsurface area, high porosity and small pore size attracting forbiomedical application3–5. Nanofiber scaffold closely mimics theextracellular matrix structure that supports cell attachment andproliferation; moreover, they can allow oxygen permeable, keepmoisture balance and protect the wounds against microorganisms.Therefore, nanofibers are excellent candidates for tissue engin-eering and functional wound-dressing material4,6,7.

Ion-exchange technology is well-known and extensivelyused in many fields such as water deionization, biologicalprocesses, food and beverage manufacture, fuel cells and

pharmaceutical applications8. Recently, much interest has focusedon ion-exchange fibers since they exhibit many advantages overion-exchange resins, for instance, the easy incorporation of largemolecules, and more rapid and efficient ion-exchange perform-ance9,10. In order to produce ion-exchange nanofibers, theelectrospinnable polymers such as polyvinyl alcohol (PVA)11

and polyethylene oxide (PEO)12 are necessary to add to thespinning solution of an ionic polymer to perform as a carrier forelectrospinning since ionic polymer alone has high conductivityand low electrospinnability. However, non-ionic polymers can beused to produce ion-exchange nanofibers via successive chemicalmodification13,14. Electrospining has known as an elegant methodto produce nanofibrous membrane with diameters ranging fromsubmicrons to nanometers. The electrospinning technique is foundto be a versatile and cost-efficient technique for producing multi-functional nanofibers3,6,15,16. Thus, electrospun nanofibers havebeen used in many fields such as biomedical sciences, filtration,optical sensors and affinity membranes17–19.

Enzyme immobilization using the adsorption method hasbeen a popular strategy for most large-scale applications due tothe ease in catalyst recycling, continuous operation and productpurification20–22. Lysozyme (EC 3.2.1.17) is an alkaline protein,which has antibacterial, antifungal, antiviral, antitumor andimmune modulatory activities23. The antimicrobial activity oflysozyme depends on its capability to possess enzymatic activityby catalyzing the hydrolysis of b-1,4 linkage between N-acetyl

Address for correspondence: Praneet Opanasopit, Faculty of Pharmacy,Silpakorn University, Nakhon Pathom 73000, Thailand. Tel: +66-34-255800. Fax: +66-34-250941. E-mail: praneet@su.ac.th; opraneet@hotmail.com

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

muramic acid and N-acetyl glucosamine residues in peptidogly-can10,24,25. Lysozyme exhibit effective antibacterial activitymainly on gram-positive bacteria as their cell wall is comprisedmuch of peptidoglycan. In contrast, gram-negative bacteria isless susceptible due to the protection of lipopolysaccharidelayers surrounding their outer membrane24–27. In this research,the poly(styrene sulfonic acid-co-maleic acid)/polyvinylalcohol (PSSA-MA/PVA) and Poly-(acrylic acid-co-maleicacid)/polyvinyl alcohol (PAMA/PVA) ion-exchange nanofibermats were fabricated and then immobilized with lysozyme todevelop the functional wound dressing material. The highporosity with small pore size and large surface area of thenanofiber mat is expected to increase the interaction betweenlysozyme and wound tissue, protect the wound and also modulatethe cellular function to promote the wound-healing process.The properties of the electrospun nanofibers and lysozyme releasecharacteristics were investigated. Moreover, the nanofiber matswere also investigated for wound healing.

Materials and methods

Materials

Lysozyme from chicken egg whites, polyvinylpyrrolidone(PVP, MW �1 300 000), PSSA-MA (sodium salt of polystyrenesulfonic acid: maleic acid¼ 3:1, average Mw¼ 20 000 g/mol)and PA-MA (50% Poly-(acrylic acid-co-maleic acid) inwater, Mw¼ 3000) were purchased from Sigma-Chemical Co.(St. Louis, MO). All other reagents and solvents were of analyticalgrade and were used without further purification.

Electrospinning of PSSA-MA/PVA and PA-MA/PVAnanofiber mats

The 0.6/1 ratio of PSSA-MA/PVA spinning solution was preparedas the previous study28. Briefly, the 20% PSSA-MA and 10% PVAaqueous solutions were mixed at solid-polymer weight ratios of0.6/1. For the PAMA/PVA spinning solution, the 50% PAMAsolution and the 10% PVA aqueous solutions were mixed at solid-polymer weight ratios of 0.2/1 to 1/1. The viscosity andconductivity of the mixed solutions were determined using aBrookfield viscometer (DV-III ultra, Brookfield EngineeringLaboratories, Middleboro, MA) and a conductivity meter(Eutech Instruments Pvt Ltd, Singapore), respectively. In theelectrospinning process, the PSSA-MA/PVA and the PAMA/PVAsolutions were contained in a glass syringe with a plain-tippedstainless steel needle with an inner diameter of 0.5 mm. Thefeeding rate was controlled at 0.4 ml/h using a syringe pump(Model NE-300, New Era Pump Systems, Farmingdale, NY). Theapplied voltage and the distance between the tip and the collectorwere fixed at 15 kV and 15 cm, respectively. The electrospunnanofibers were collected on aluminum foil that was placed on arotating collector. The electrospinning process was conducted atroom temperature (25 �C).

Crosslinking process

The PSSA-MA/PVA and the PAMA/PVA nanofiber mats wereplaced in a hot-air oven at 130 �C for 5 h to induce crosslinking.

Determining of equilibrium time for lysozyme adsorption

To determine the equilibrium time for lysozyme adsorption,the lysozyme was loaded on the PSSA-MA/PVA and the PAMA/PVA nanofiber mats by immersing the nanofiber mats intolysozyme solution (250 mg/ml in phosphate buffer; pH 9) andallowing the mixtures to stir on a magnetic stirrer. At pre-determining time of 0, 2, 6 and 8 h, an aliquot of the solution

was withdrawn. The amount of lysozyme loaded into each fibermat was determined by calculating the difference between theamount of lysozyme in the initial loading solution and theamount in the collected post-loading and washing solutions, asdetermined by HPLC. The lysozyme-loaded nanofiber matswere washed several times with deionized water and dried at37 �C overnight in a hot air oven. The lysozyme-loaded nanofibermats were kept in a tightly sealed container at 4–8 �C.

Loading of lysozyme on nanofibers and lysozyme content

After known the equilibrium time for lysozyme immobiliza-tion, the lysozyme was loaded on the PSSA-MA/PVA and thePAMA/PVA nanofiber mats by the same method as previousmentioned for 6 h. The concentration of lysozyme solution beforeand after loading was examined using HPLC (AgilentTechnology, Santa Clara, CA). A VertiSep� AQS C18 column(250� 4.6 mm, 5mm particle size) with a C18 guard column wasused. The elution was carried out with gradient solvent systemsconsisting of 1% acetonitrile, 0.2% trifluoroacetic acid and 98.8%water (mobile A) and 70% acetonitrile, 0.2% trifluoroacetic acidand 29.8% water (mobile B) with a flow rate of 1 ml/min atambient temperature. The gradient was programmed as follows:100% A for 0–3 min, constant at 45% A for 9 min, 45% A to 20%A in 3 min and 20% A to 100% A in 5 min. The wavelengths ofthe spectrofluorimetric detector were set at Ex¼ 276 nmand Em¼ 345 nm29. The amount of lysozyme immobilizededinto each fiber mat was determined by calculating asmentioned above.

Characterization of lysozyme loaded nanofibers

Scanning electron microscope

The morphology and diameter of the ion-exchange nanofibermats and lysozyme immobilizeded ion-exchange nanofibermats were examined using a scanning electron microscope(SEM, Camscan M� 2000, Cambridge, UK). For this process,a small section of the fiber was sputtered with a thin layer ofgold prior to the SEM observations. The average diameter ofthe fibers was determined using image analysis software(JMicroVision V.1.2.7, Geneva, Switzerland).

Fourier transform infrared spectrophotometry

The chemical structure of the ion-exchange nanofiber matswas characterized using a Fourier transform infrared spectropho-tometer (FT-IR, Nicolet 4700, Minnesota, MN). The fibersamples were ground and pressed into KBr dishes pellets beforebeing analyzed by the FT-IR analysis from 400 to 4000 cm�1.

Powder X-ray diffractometry

Powder X-ray diffraction analyses (PXRD, Miniflex II, Rigaku,Japan) of the samples were carried out to investigate the physicalstate of the lysozyme on the nanofiber mats using Nickel-filteredCu radiation generated in a sealed tube operated at 30 kv and15 mA. The diffraction patterns of the nanofiber mats wererecorded in the � range of 5–45� at 5� min�1.

In vitro release

The in vitro release studies were investigated. Briefly, 5 mg of thelysozyme-immobilized nanofiber mats was placed in a 10 mlbottle containing 3 ml of phosphate buffer (pH 7.4) that wasincubated at 37 �C while stirring at 100 rpm. To determine theamount of lysozyme released from the ion-exchange nanofibermats after a given interval, an aliquot (500 mL) of the releasemedium solution was withdrawn and replaced with the same

2 P. Tonglairoum et al. Pharm Dev Technol, Early Online: 1–8

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

volume of fresh medium to maintain a constant volume. Theamounts of lysozyme in the sample solutions were analyzed byHPLC. The experiments were conducted in triplicate.

Lysozyme activity assay

The biological activities of the lysozyme immobilized PSSA-MA/PVA and the PAMA/PVA ion-exchange nanofiber matswere examined using an EnzChek lysozyme assay kit (E-22013).M. lysodeikticus cells were employed as a substrate. The sampleswere prepared by elution the lysozyme from the nanofiber matsin 2 M KCl (3 mL) at room temperature and continuously stirredfor 24 h. A 50mL of the solution after eluting was subsequentlymixed with 50 mg/mL (50 mL) of M. lysodeikticus, labeled withfluorescein in a 96-well plate. The mixtures were incubated at37 �C for 30 min, and the fluorescence intensity of each samplewas measured in a microplate reader (Universal MicroplateAnalyzer, Model AOPUS01 and AI53601, Packard BioScience,CT). The fluorescence absorption maxima and emission maximaof the digested products from the substrate were at 485 and535 nm, respectively. Specific activity was defined in terms ofunits of activity per milligram of protein (U/g). All samples wereassayed in triplicate.

Wound healing test

The wound healing study was approved by an InvestigationalReview Board (Animal Studies Ethics Committee, Faculty ofPharmacy, Silpakorn University, Approval No.2/2557). In thisstudy, male Wistar rats (310–360 g) were used. After anesthetiza-tion, two full-thickness rectangular wounds with a surfacearea of 0.8 cm2 were produced on the neck area of the dorsalside of each rat. The wound was covered with a nanofibermat equal size to its size, gauze and commercial antibacterial gauzedressing (Bactigras�, Smith & Nephew, London, UK) (n¼ 6). Thearea of the wound was measured every day using the planimetrymethod until the wound completely healed. The percentage ofwound healing is defined by Equation (1).

% Wound area¼ A=Aið Þ � 100 ð1Þ

where Ai is the initial wound area, and A is the wound area after afixed time interval.

Statistical analysis

All experimental measurements were collected in triplicate. Thevalues are expressed as the mean ± standard deviation (SD).The statistical significance of the differences in each experimentwas examined using one-way analysis of variance (ANOVA),followed by a least significant difference (LSD) post hoc test. Thedifferences were significant at p50.05.

Results and discussion

PSSA-MA/PVA and PAMA/PVA nanofiber mats

The PAMA/PVA nanofibers were electrospun from thespinning solutions with various PAMA/PVA ratios, includingratios of 0.2/1, 0.4/1, 0.6/1 and 0.8/1 (g/g) and subsequent thermalcrosslinked. The solution parameters before electrospinningare listed in Table 1. As the increasing the amounts of PAMAin the spinning solution, the viscosity and conductivityincreased. After being electrospun, bead free nanofibers wereobtained. The amount of PAMA in the spinning solution affectedthe morphology of the nanofiber mats. When the ratio of PAMAwas increased from 0.2/1 to 0.8/1, the average diameter of thenanofibers increased from 394 ± 76 nm to 719 ± 187 nm and

the size distribution was also increased. These may be due to theincrease in viscosity of spinning solution when the amount ofPAMA/PVA increased. Solution properties have been establishedto affect the morphology of the fiber. One of the major parameterinfluencing the fiber diameter is solution viscosity. Actually,the solution viscosity is regarding to the extent of polymermolecule chains entanglement within the solution. A higherviscosity results in a larger fiber diameter30,31. At a high ratioof PAMA/PVA, rough surface fibers were observed, asillustrated in Figure 1.

The PSSA-MA/PVA ion-exchange nanofibers at the poly-mer weight ratio of 0.6:1 with smooth fiber were alsoprepared using electrospinning process as previously describe28.The average diameter of the nanofibers was 321.69 ± 47.4 nm.The PSSA-MA/PVA and PAMA/PVA nanofiber at the weightratio of 0.6:1 and 0.8:1, respectively, were selected for cross-linking process.

After electrospinning process, the PSSA-MA/PVA and thePAMA/PVA nanofiber mats were placed in a hot-air oven at130 �C for 5 h to induce crosslinking as previously describe28. Thewater insolubilization and FTIR spectra analyzing were usedto determine the successful crosslinking of the fibers. In caseof uncrosslinked PAMA/PVA nanofibers, the percentage ofwater insolubilization could not be determined since the fiberscompletely dissolved during the investigation; the crosslinkedPAMA/PVA nanofibers achieved the water insolubilizationpercentage of 95.7%. Figure 2 presents the FT-IR spectra ofPAMA/PVA and PSSA-MA/PVA nanofiber before and aftercrosslinked. The spectrum of PSSA-MA/PVA nanofibers exhib-ited the sulfonic acid peak of PSSA at 1174, 1137 and 676 cm�1,the O–H stretching vibration peak at 3342 cm�1. The C¼Ovibration peak and the O–H peak of maleic acid at 1704and 921 cm�1, respectively. The crosslinked PSSA-MA/PVAnanofibers showed the different peak in comparison to theun-crosslinked nanofibers. The O–H peak of maleic acid at921 cm�1 disappeared due to the ester bond creation from COOHgroups of maleic acid and OH groups of PVA. The C¼O peakof ester bond occurred at 1704 cm�1 but the C–O peak of esterbond at 1200 cm�1 was hidden by the sulfonic acid peak. Theseresults are in accordant with our previous study which reportedthe crosslinking of PSSA/PVA nanofiber28. The spectrum ofPAMA/PVA exhibited a strong absorption band at 1720 which istypical of carbonyl (–C¼O) stretching of COOH group.Absorption bands at 1459, 1404 assigned to –COO stretchingvibration. Peak at 2927 and 2851 indicate –OH stretchingvibration. The crosslinked PAMA/PVA nanofibers showed littledifference of peak compared with the un-crosslinked nanofibers.The increasing in an intensity of the C¼O peak of the ester bondat 1181 cm�1 could be a result from the interaction betweenCOOH groups of acrylic acid or maleic acid with the OH groupsof PVA to create ester bond. These results suggest that the fiberswere crosslinked after thermal treatment.

Table 1. Solution parameters before electrospining of PAMA/PVA andPSSA-MA solution.

NanofiberPolymer

weight-ratio Viscosity (cP) Conductivity (mS)

PAMA/PVA 0.2/1 785.7 ± 0.74 0.994 ± 0.25PAMA/PVA 0.4/1 879.1 ± 2.09 1.565 ± 0.48PAMA/PVA 0.6/1 1018.3 ± 1.53 1.983 ± 0.26PAMA/PVA 0.8/1 1193.7 ± 2.82 2.450 ± 0.47PSSA/PVA 0.6/1 861.4 ± 1.75 8.910 ± 0.55

Each value represents the mean ± SD from three independent.

DOI: 10.3109/10837450.2014.954726 Lysozyme immobilized ion-exchange nanofiber mats 3

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

Equilibrium time for lysozyme adsorption

To determine the equilibrium time for lysozyme adsorption,the lysozyme was loaded on the PSSA-MA/PVA and the PAMA/PVA nanofiber mats by immersing the nanofiber mats intolysozyme solution and allowing the mixtures to stir on a magneticstirrer and determined the amount of lysozyme loaded into thenanofiber after 0, 2, 6 and 8 h. The results indicate that lysozymewas gradually absorbed into the PSSA-MA/PVA ion-exchangenanofiber with higher extend than that is absorbed on the PAMA/PVA ion-exchange nanofiber. These may be related to theinteraction between lysozyme and anionic groups of the ion-exchange nanofiber. The PSSA-MA/PVA nanofiber contained bothstrong (SO�3 ) and weak (COO�) anionic groups while the PAMA/PVA contained just only the weak anionic groups (COO�). Thus,the PSSA-MA/PVA nanofibers have greater interaction withlysozyme in comparison to the PAMA/PVA nanofiber. Thus,lysozyme can be absorbed on PSSA-MA/PVA nanofiber in higheramount than PAMA/PVA nanofiber. The dynamic equilibrium forlysozyme absorption on the PSSA-MA/PVA nanofiber mats and

the PAMA/PVA nanofiber mats were reached at 0.5 and 6 h,respectively, as showed in Figure 3. Thus, these time points wereselected for immobilized lysozyme on the nanofibers.

Characterization of lysozyme-loaded nanofiber

Scanning electron microscope

The lysozyme was immobilized on the PSSA-MA/PVA and thePAMA/PVA nanofiber mats by immersing the ion-exchangenanofiber mats into lysozyme solution. Figure 4 representsthe morphology of the PAMA/PVA and PSSA-MA/PVAion-exchange nanofiber mats before and after immobilized withlysozyme. Both ion-exchange nanofibers became flatter aftersoaking in the lysozyme solution.

Fourier transform infrared spectrophotometry

After lysozyme being immobilized on the PAMA/PVA and PSSA-MA/PVA nanofibers, the FT-IR spectra of lysozyme-immobilizedPAMA/PVA and lysozyme-immobilized PSSA-MA/PVA

Figure 1. The SEM image (1,000x) ofPAMA/PVA ion-exchange nanofiber matswith different PAMA/PVA weight ratios:(a) 0.2/1, (b) 0.4/1, (c) 0.6/1 and (d) 0.8/1.

Figure 2. FT�IR spectra of PAMA/PVA andPSSA-MA/PVA ion-exchange nanofiberbefore and after thermally crosslinked.

4 P. Tonglairoum et al. Pharm Dev Technol, Early Online: 1–8

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

nanofibers were changed from the spectra of the blank fibers. Asshown in Figure 5, it is clear that there is a significant shift in majorpeaks which indicates the interaction between the polymer andlysozyme.

Powder X-ray diffractometry

The physical status of lysozyme on the PAMA/PVA ion-exchangenanofiber mats and the PSSA-MA/PVA ion-exchange nanofibermats were investigated using a powder X-ray diffractometer.The powder X-ray diffraction patterns of the lysozyme powder,blank PAMA/PVA and PSSA-MA/PVA ion-exchange nanofibermats and lysozyme-immobilized ion-exchange nanofiber matsare presented in Figure 6. No such peak was found in thediffractograms of both lysozyme-loaded ion-exchange nanofibermats indicating that lysozyme was immobilized into the nanofibermats in an amorphous state.

Lysozyme content and activity

In this study, the lysozyme was immobilized on the PSSA-MA/PVA and the PA-MA/PVA ion-exchange nanofiber mats by

Figure 4. The SEM image (1000�) of(a) PAMA/PVA nanofiber, (b) lysozymeimmobilized PAMA/PVA nanofiber,(c) PSSA-MA/PVA nanofiber and(d) lysozyme immobilized PSSA-MA/PVAnanofiber.

Figure 5. FT�IR spectra of PAMA/PVA andPSSA-MA/PVA ion-exchange nanofiberbefore and after immobilized with lysozyme.

Figure 3. The dynamic equilibrium for lysozyme absorption on thePSSA-MA/PVA nanofiber mats and the PAMA/PVA nanofiber mats.

DOI: 10.3109/10837450.2014.954726 Lysozyme immobilized ion-exchange nanofiber mats 5

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

adsorption method. The PSSA-MA/PVA nanofiber consists ofboth strong (SO�3 ) and weak (COO�) anionic groups, and thePAMA/PVA contained weak anionic groups (COO�) which caninteract with the lysozyme. The total content of lysozyme in thelysozyme-immoblized PSSA-MA/PVA and PAMA/PVA nanofi-ber mats was determined using the indirect method. The lysozymeactivity of the lysozyme immobilized nanofiber mats was alsomeasured to confirm the presence of lysozyme on the nanofibermats and the possibility to use the nanofiber mats for woundhealing application. The biological activities of the lysozyme-immobilized PSSA-MA/PVA and the PAMA/PVA ion-exchangenanofiber mats were examined with an EnzChek lysozyme assaykit (E-22013). The lysozyme content and activity of lysozyme-immobilized PSSA-MA/PVA and PAMA/PVA nanofiber mats arepresented in Table 2. Lysozyme could be adsorbed on the PSSA-MA/PVA nanofiber mats in higher amount due to the strongerstrength of the anionic group on the PSSA-MA/PVA nanofiber(SO�3 ) in comparison to the PAMA/PVA nanofiber mats (COO�).In addition, the lysozyme activity is related to the lysozymecontent. The lysozyme-immobilized PSSA-MA/PVA nanofiberexhibited higher lysozyme activity than the lysozyme-immobi-lized PAMA/PVA nanofiber mats.

Figure 8. The wound healing on day 1, 4, 8and 11 after treatment with (a) lysozyme-immobilized PSSA-MA/PVA nanofiber mats,(b) lysozyme-immobilized PAMA/PVAnanofiber mats, (c) sterile gauze (negativecontrol) and (d) commercial antibacterialgauze (Bactigras�, positive control).

Figure 6. X-ray diffraction pattern of the PAMA/PVA and PSSA-MA/PVA nanofiber before and after immobilized with lysozyme and X-raydiffraction pattern of pure lysozyme powder.

Figure 7. Release profiles of lysozyme from (˙) lysozyme-immobilizedPAMA/PVA and (g) lysozyme-immobilized PSSA-MA/PVA nanofiber.

Table 2. Total lysozyme content and activity of lysozyme-immobilizedPSSA-MA/PVA and PAMA/PVA ion-exchange nanofibers.

Nanofiber matsLysozyme

content (mg/g)Lysozyme

activity (IU/g)

PSSA-MA/PVA 165.9 ± 11.6 17.88 ± 1.03PAMA/PVA 647.6 ± 58.8 7.62 ± 1.24

Each value represents the mean ± SD from three independent.

6 P. Tonglairoum et al. Pharm Dev Technol, Early Online: 1–8

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

In vitro release

In vitro release of the lysozyme from the lysozyme-immobilizedPSSA-MA/PVA and lysozyme-immobilized PAMA/PVA nanofi-ber mats were investigated in phosphate buffer (pH 7.4). In thiscase, the electrolyte contained in phosphate buffer underwent theexchange reaction with lysozyme and the lysozyme released tothe release medium. The release behavior of lysozyme from theion-exchange nanofiber mats are exhibited in Figure 7. Thelysozyme was rapidly released and released in the similar mannerfrom the PAMA/PVA ion-exchange nanofibers and PSSA-MA/PVA ion-exchange nanofibers. The percentage of cumulativelysozyme release within 1 h was 49 and 51% from lysozyme-immobilized PAMA/PVA ion-exchange nanofibers and lysozyme-immobilized PSSA-MA/PVA ion-exchange nanofibers, respect-ively. This result reveals that the lysozyme was burst releasedfrom the nanofiber mats. Previous studies also reported the rapidrelease of neomycin and salicylic acid at low concentration fromion-exchange fiber32,33. However, exchange reaction is dependedon the amount and valence of the electrolytes in the externalsolution.

Wound healing test

In the wound healing study, two wounds with areas of 0.8 cm2

were produced on the neck area of the dorsal side of each rat. Theimages of the wound healing on day 1, 4, 8 and 11 after treatmentwith sterile gauze (negative control), lysozyme-immobilizedPSSA-MA/PVA, lysozyme-immobilized PAMA/PVA nanofibermats and commercial antibacterial gauze (Bactigras�, positivecontrol) are depicted in Figure 8. In all treatment groups, thewound was recuperated within 11 days after intervention. Thewound areas were decreased gradually and hit approximately 0%after 11 days as presented in Figure 9. The lysozyme-immobilizedPSSA-MA/PVA nanofiber mats and lysozyme-immobilized PA-MA/PVA nanofiber mats exhibited significantly faster healingrate than gauze on day 1–5 and on day 3–5, respectively (p50.05)and similar to the commercial antibacterial gauze dressing. Thismay be due to the release of lysozyme from the nanofiber mats tothe wound area that hasten the healing process by deactivatebacterias34. After that, the % wound area of all treatments sinceDay 6 and recovery was similar. This suggests that wound healingwas processed by a body mechanism, independent from theeffects of the treatment35.

Conclusion

Lysozyme-immobilized ion-exchange nanofibers were success-fully fabricated using electrospinning and crosslinking process,followed by adsorption immobilization of lysozyme. The lyso-zyme is rapidly released from both the PAMA/PVA ion-exchangenanofibers and PSSA-MA/PVA ion-exchange nanofibers, whichretains antibacterial activity, and accelerates the wound healingprocess. Thus, these antibacterial electrospun nanofiber mats havepromising potential for use as effective wound dressings.

Declaration of interest

None declared. The authors would like to acknowledge the Commissionof Higher Education (Thailand) and the Thailand Research Fundsthrough the Royal Golden Jubilee Ph.D. Program (Grant No.PHD/0092/2554) and the Silpakorn University Research and development institute(Grant No.SURID56/02/01).

References

1. Serincay H, Ozkan S, Yılmaz N, et al. PVA/PAA-based antibacterialwound dressing material with Aloe Vera. Polym Plast Technol Eng2013;52:1308–1315.

2. Jayakumar R, Prabaharan M, Sudheesh Kumar PT, et al.Biomaterials based on chitin and chitosan in wound dressingapplications. Biotechnol Adv 2011;29:322–337.

3. Tonglairoum P, Chuchote T, Ngawhirunpat T, et al. Encapsulationof plai oil/2-hydroxypropyl-b-cyclodextrin inclusion complexes inpolyvinylpyrrolidone (PVP) electrospun nanofibers for topicalapplication. Pharm Dev Technol 2014;19:430–437.

4. Nguyen TTT, Ghosh C, Hwang S, et al. Characteristics of curcumin-loaded poly (lactic acid) nanofibers for wound healing. J Mater Sci2013;48:7125–7133.

5. Tonglairoum P, Ngawhirunpat T, Rojanarata T, et al. Fast-actingclotrimazole composited PVP/HPbCD nanofibers for oralCandidiasis application. Pharm Res 2014 [Epub ahead of print].

6. Sill TJ, Von Recum HA. Electrospinning: applications in drugdelivery and tissue engineering. Biomaterials 2008;29:1989–2006.

7. Liang D, Hsiao BS, Chu B. Functional electrospun nanofibrousscaffolds for biomedical applications. Adv Drug Deliv Rev 2007;59:1392–1412.

8. An H, Shin C, Chase GG. Ion exchanger using electrospunpolystyrene nanofibers. J Mem Sci 2006;283:84–87.

9. Hanninen K. Characterization of ion-exchange fibers for controlleddrug delivery [dissertation]. Finland: University of Helsinki; 2008.

10. Conte A, Buonocore GG, Sinigaglia M, Del Nobile MA.Development of immobilized lysozyme based active film. J FoodEng 2007;78:741–745.

11. Matsumoto H, Yako H, Minagawa M, Tanioka A. Characterizationof chitosan nanofiber fabric by electrospray deposition: electrokin-etic and adsorption behavior. J Colloid Interface Sci 2007;310:678–681.

12. Seo H, Matsumoto H, Hara S, et al. Preparation of polysaccharidenanofiber fabrics by electrospray deposition: additive effects ofpoly(ethylene oxide). Polym J 2005;37:391–398.

13. Matsumoto H, Wakamatsu Y, Minagawa M, Tanioka A. Preparationof ion exchange fiber fabrics by electrospray deposition. J ColloidInterface Sci 2006;293:143–150.

14. Park HJ, Na CK. Preparation of anion exchanger by amination ofacrylic acid grafted polypropylene nonwoven fiber and its ion-exchange property. J Colloid Interface Sci 2006;301:46–54.

15. Uyar T, Besenbacher F. Electrospinning of cyclodextrin functiona-lized polyethylene oxide (PEO) nanofibers. Eur Polym J 2009;45:1032–1037.

16. Bai J, Yang Q, Li M, et al. Synthesis of poly (N-vinylpyrrolidone)/b-cyclodextrin composite nanofibers using electrospinning tech-niques. J Mater Process Technol 2008;208:251–254.

17. Yao LD, Tang Y, Huang ZF. Nicotinic acid voltammetric sensorbased on molecularly imprinted polymer membrane-modified elec-trode. Anal Lett 2007;40:677–688.

18. Haupt K, Mosbach K. Molecularly imprinted polymers and their usein biomimetic sensors. Chem Rev 2000;100:2495–2504.

Figure 9. Wound healing tests of (˙) sterile gauze (negative control),(g) commercial antibacterial gauze (Bactigras�, positive control),(�) lysozyme-immobilized PAMA/PVA nanofiber mats and (m) lyso-zyme-immobilized PSSA-MA/PVA nanofiber mat. Difference values*were statistically significant (p50.05) compared with gauze. The dataare presented as mean ± SD (n¼ 6).

DOI: 10.3109/10837450.2014.954726 Lysozyme immobilized ion-exchange nanofiber mats 7

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.

19. Kriz O, Ramstrom O, Mosbach K. Molecular imprinting: newpossibilities for sensor technology. Anal Chem 1997;69:345A–349A.

20. Ozturk N, Akgol S, Arisoy M, Denizli A. Reversible adsorption oflipase on novel hydrophobic nanospheres. Sep Purif Technol 2007;58:83–90.

21. Sanjay G, Sugunan S. Fixed bed reactor performance of invertaseimmobilized on montmorillonite. Catal Commun 2006;7:1005–1011.

22. Sari M, Akgol S, Karatas M, Denizli A. Reversible immobilizationof catalase by metal chelate affinity interaction on magnetic beads.Ind Eng Chem Res 2006;45:3036–3043.

23. Samaranayake YH, Samaranayake LP, Pow EH, et al. Antifungaleffects of lysozyme and lactoferrin against genetically similar,sequential Candida albicans isolates from a HumanImmunodeficiency Virus-infected Southern Chinese cohort. J ClinMicrobiol 2001;39:3296–3302.

24. Losso JN, Nakai S, Charter EA. Natural food antimicrobial systems.In: Naidu AS, ed. Lysozyme. Boca Raton (FL): CRC Press LLC;2000:185–210.

25. Barbiroli A, Bonomi F, Capretti G, et al. Antimicrobial activity oflysozyme and lactoferrin incorporated in cellulose-based foodpackaging. Food Control 2012;26:387–392.

26. Saravanan R, Shanmugam A, Ashok P, et al. Studies on isolation andpartial purification of lysozyme from egg white of the lovebird(Agapornis species). Afr J Biotechnol 2009;8:107–109.

27. Lian ZX, Ma ZS, Wei J, Liu H. Preparation and characterization ofimmobilized lysozyme and evaluation of its application in ediblecoatings. Process Biochem 2012;47:201–208.

28. Nitanan T, Akkaramongkolporn P, Rojanarata T, et al. Thermallycrosslinkable poly (styrene sulfonic acid-co-maleic acid) (PSSA-MA)/polyvinyl alcohol (PVA) ion exchange fibers. Polym Bull2013;70:1431–1444.

29. Riponi C, Natali N, Chinnici F. Quantification of hen’s egg whitelysozyme in wine by an improved HPLC-FLD analytical method.Am J Enol Viticult 2007;58:405–409.

30. Uslu I, Keskin S, Gul A, et al. Preparation and properties ofelectrospun poly(vinyl alcohol) blended hybrid polymer with Aloevera and HPMC as wound dressing. Hacettepe J Biol Chem 2010;38:19–25.

31. Ramakrishna S, Fujihara K, Teo WE, et al. An introduction toelectrospinning and nanofibers. 1st ed. New Jersey: WorldScientific; 2005:91–101.

32. Nitanan T, Akkaramongkolporn P, Rojanarata T, et al. Neomycin-loaded poly (styrene sulfonic acid-co-maleic acid)(PSSA-MA)/polyvinyl alcohol (PVA) ion exchange nanofibers for wounddressing materials. Int J Pharm 2013;448:71–78.

33. Hanninen KR, Murtomaki LS, Kaukonen AM, Hirvonen JT.The effect of valence on the ion-exchange process: theoreticaland experimental aspects on compound binding/release. J Pharm Sci2007;96:117–131.

34. Mecitoflu C, Yemenicioflu A, Arslanoflu A, et al. Incorporationof partially purified hen egg white lysozyme into zeinfilms for antimicrobial food packaging. Food Res Int 2006;39:12–21.

35. Chen JP, Chang GY, Chen JK. Electrospun collagen/chitosannanofibrous membrane as wound dressing. Colloid Surf APhysicochem Eng Aspects 2008;313–314:183–188.

8 P. Tonglairoum et al. Pharm Dev Technol, Early Online: 1–8

Phar

mac

eutic

al D

evel

opm

ent a

nd T

echn

olog

y D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

of

Con

nect

icut

on

10/1

0/14

For

pers

onal

use

onl

y.