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Biomaterials 32 (2011) 9557e9567

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Lysostaphin-functionalized cellulose fibers with antistaphylococcalactivity for wound healing applications

Jianjun Miaoa,f,g,1, Ravindra C. Pangulea,f,g,1, Elena E. Paskalevaa,f, Elizabeth E. Hwangh,Ravi S. Kanea,f,g,*, Robert J. Linhardta,b,c,d, f,g,*, Jonathan S. Dordicka,c,d,e, f,g,*aDepartment of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAbDepartment of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAcDepartment of Biology, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAdDepartment of Biomedical Engineering, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAeDepartment of Materials Science and Engineering, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAfCenter for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAgRensselaer Nanotechnology Center, Rensselaer Polytechnic Institute, 110, 8th St, Troy, NY 12180, USAhDepartment of Chemistry, Williams College, Williamstown, MA 01267, USA

a r t i c l e i n f o

Article history:Received 9 July 2011Accepted 26 August 2011Available online 28 September 2011

Keywords:AntimicrobialBiocompatibilityEnzymeCelluloseKeratinocyteIn vitro test

* Corresponding authors. Department of ChemicalRensselaer Polytechnic Institute, 110, 8th St, Troy, NY2899; fax: þ1 518 276 2207.

E-mail addresses: kaner@rpi.edu (R.S. Kane), lindordick@rpi.edu (J.S. Dordick).

1 Authors contributed equally to this work.

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.08.080

a b s t r a c t

With the emergence of “super bacteria” that are resistant to antibiotics, e.g., methicillin-resistantStaphylococcus aureus, novel antimicrobial therapies are needed to prevent associated hospitalizationsand deaths. Bacteriophages and bacteria use cell lytic enzymes to kill host or competing bacteria,respectively, in natural environments. Taking inspiration from nature, we have employed a cell lyticenzyme, lysostaphin (Lst), with specific bactericidal activity against S. aureus, to generate anti-infectivebandages. Lst was immobilized onto biocompatible fibers generated by electrospinning homogeneoussolutions of cellulose, cellulose-chitosan, and cellulose-poly(methylmethacrylate) (PMMA) from 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), room temperature ionic liquid. Electron microscopicanalysis shows that these fibers have submicron-scale diameter. The fibers were chemically treated togenerate aldehyde groups for the covalent immobilization of Lst. The resulting Lst-functionalizedcellulose fibers were processed to obtain bandage preparations that showed activity against S. aureusin an in vitro skin model with low toxicity toward keratinocytes, suggesting good biocompatibility forthese materials as antimicrobial matrices in wound healing applications.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Super bacteria, resistant to nearly all currently prescribed anti-biotics, can result from the overuse or improper use of antibiotics[1]. One such bacterium, methicillin-resistant Staphylococcusaureus (MRSA), is resistant tomost commercially available b-lactamtype of antibiotics including, but not limited to, methicillin,oxacillin, penicillin, and amoxicillin [2]. As a result, MRSA causednearly a half million hospitalizations and resulted in approximately11,000 deaths in the U.S. in 2005 [3]. The healthcare costs associ-ated with MRSA-related infections exceed 14 billion dollars per

and Biological Engineering,12180, USA. Tel.: þ1 518 276

har@rpi.edu (R.J. Linhardt),

All rights reserved.

year [4]. Although life-threatening MRSA is found mostly inhospital settings, there has been an increase in the number of casesof MRSA infections caused in community settings, resulting indiseases, such as abscesses, endocarditis, toxic shock syndrome andsepsis. Among the most common causes of community-associatedMRSA infections is skin infection through burns and wounds [4].

A thorough understanding of MRSA infections is needed todesign effective treatments, appropriate prevention strategies, andto develop new classes of antibiotics [5]. Efforts to control thespread of MRSA require the design and synthesis of effectivealternatives to antibiotics, including anti-bacterial matrices eitherin the form of coatings, surface wipes or bandages. Manynon-specific chemical agents including silver [6e8], hydrogenperoxide [9e11], iodine [12,13], and polypeptides and polycations[14e16], have been incorporated into such matrices to kill bacterialpathogens. However, some of these current approaches havecertain disadvantages. For example, continuous release of lowmolecular weight anti-bacterial agents from matrices can lead to

J. Miao et al. / Biomaterials 32 (2011) 9557e95679558

diminishing effectiveness over time [17]. Also, the cytotoxicity ofsome anti-bacterial agents, such as silver that binds DNA andprevents replication, adversely impacts the promise of these non-specific, low molecular weight agents in treating infections[18,19]. Silver, hydrogen peroxide and iodine, while useful againstbroad range of bacterial targets, can cause irritation and inflam-mation [20e22]. In the case of polycationic polymer-based coat-ings, the surface may need to be washed with cationic surfactant tofacilitate removal of the attached cells or cell debris and to recoverantimicrobial activity [23]. Moreover, the common use of broadly-active antimicrobial agents (e.g., antibiotics) has led to the emer-gence of resistance in commonly found non-pathogenic microor-ganisms. Altogether, these factors providemotivation to investigatealternative antimicrobial agents that are benign and species-specific.

In nature, bacteriophages and bacteria employ cell-lyticenzymes to specifically target their hosts or to overgrow in thepresence of competing microbial species. Lysostaphin (Lst), forexample, a cell lytic enzyme secreted by S. simulans, is highlyspecific and effective against S. aureus. Lst acts by cleaving thepentaglycine cross-bridges in the peptidoglycan layer of staphylo-coccal cell walls [24]. Taking advantage of its antistaphylococcalactivity, Lst has been used to treat neonatal infection in a rat model[25], to disrupt biofilms [26], and to prepare antistaphylococcalpaint nanocomposites [24]. Regarding the latter, we previouslydemonstrated that Lst, when conjugated with oxidized multi-wallcarbon nanotubes and incorporated into a paint matrix, showedcontact-mediated killing of w99.9% MRSA cells within 2 h. Whilecarbon nanotube conjugation leads to high enzyme loading, thepotential cytotoxicity of carbon nanotubes reduces their potentialapplication as a bandage matrix [27,28], motivating the develop-ment of alternative non-cytotoxic nanoscale supports.

Fiber-based wound dressings conjugated with active anti-bacterial agents, can be prepared using electrospinning. Electro-spinning produces micro- to nanoscale fibers having very highspecific surface areas, ranging from 10 to 1000 m2/g, and highporosities of up to 80% [29]. Many synthetic polymers and naturalbiopolymers [29,30] can be electrospun into nanofibers. Poly(L-lactic acid) (PLLA) is a frequently used non-cytotoxic and biode-gradable polymer in preparing electrospun fibers for drug deliveryand tissue engineering applications [31e34]. However, the surfacemodification of PLLA fiber for bioconjugation poses a major chal-lenge [35]. Electrospun fiber mats prepared from other non-cytotoxic polymers such as poly(vinyl alcohol), collagen [36],dextran [37], or hyaluronic acid [38] require additional treatmentsteps, such as heat curing or cross-linking to avoid the loss of fiberstructure through gel formation on contact with aqueous media.When considering appropriate materials for fiber-based wounddressings with expected exposure to wound exudates, additionalfactors must be addressed, including wettability, fluid affinity andbiopolymer stability.

Polysaccharides are natural biopolymers that show excellentbiocompatibility properties and allow for easy chemical modifica-tion [39e42]. Cellulose, the most abundant renewable naturalbiopolymer is a linear polysaccharide consisting of D-glucose resi-dues linked by b-(1/4)-glycosidic bonds. It is often used in pack-aging, textiles, and biomedical devices because of itsbiodegradability, biocompatibility, and regenerability [43]. Cellu-lose has also been successfully electrospun into microfibers andnanofibers using room temperature ionic liquids (RTILs) [43,44]. Oncontact with aqueous media, cellulose fibers do not form gels ordissolve, but instead become hydrated, thus serving as a well-accepted material for bandages [45]. Additionally, the diameterand porosity of electrospun cellulose fibers can easily be optimizedfor wound dressing applications.

In the current work, we generated three different cellulosefibers by electrospinning solutions of cellulose, cellulose-chitosan,and cellulose-PMMA prepared in an RTIL. Different chemicalmodifications were performed to generate enzyme-reactive fibers.The Lst-functionalized cellulose fibers were characterized andtested for their antimicrobial activity against S. aureus to obtain anoptimal preparation with stable bioconjugation and high antimi-crobial activity. Biocompatibility of Lst-cellulose fiber mats wasconfirmed by testing their cytotoxicity against relevant skin cells(HaCaT keratinocyte cells). Finally, a skin-like model constitutingmonolayer culture of keratinocytes was used to assess anti-staphylococcal activity of Lst-cellulose preparation and to mimica scenario where a skin infection is treated with an Lst-cellulosefiber mat.

2. Materials and methods

2.1. Materials

1-Ethyl-3-methylimidazolium acetate ([EMIM][OAc]) and 5 wt% cellulose(degree of cellulose polymerization 1100) in [EMIM][OAc] (Cellionic�) wereobtained from SigmaeAldrich (St. Louis, Missouri, USA). Medium molecular weightchitosan with viscosity of 800 cP was purchased from SigmaeAldrich. PMMA withmolecular weight of 120 kDa and inherent viscosity of 0.2 was also obtained fromSigmaeAldrich. Solvents were purchased from either Fisher Scientific or Sigma-eAldrich unless specified.

2.2. Preparation of the solution for electrospinning cellulose, cellulose-chitosan andcellulose-PMMA

The cellulose solutionwas prepared by diluting the 5 wt% cellulose [EMIM][OAc]solution to 1.75 wt% with neat [EMIM][OAc]. Solutions (1.5% and 2.0%) were alsoprepared to optimize fiber thickness. The mixture was then mechanically stirredusing a magnetic stirrer at 80 �C to form a homogeneous solution.

The cellulose-chitosan solutions were prepared following a similar method asdescribed above, except with addition of DMSO. Specifically, 3 g of 5% cellulose in[EMIM][OAc] was mixed with 3.75 g of [EMIM][OAc] at 80 �C to give a homogenoussolution. The diluted cellulose solution was further mixed with 0.75 g of DMSO,followed by addition of 30 mg medium molecular weight chitosan to give a finalsolution containing 2.0% (w/w) cellulose and 0.4% (w/w) chitosan.

The preparation of cellulose-PMMA solutionwas different from that of cellulose-chitosan due to the hydrophobic nature of PMMA. For example, 1 g of PMMA wasdissolved in 5 g of DMF followed by addition of 15 g DMSO to give a 5% PMMA stocksolution, from which 2 g solution was mixed with 8 g of 5% cellulose solution and7.8 g of [EMIM][OAc]. Triton X100 (100 ml) was added to the resultingmixture, whichwas stirred at 80 �C for 1 h to yield a homogenous solution. The resulting solutioncontained 2.25% (w/w) cellulose and 0.5% (w/w) PMMA.

2.3. Electrospinning cellulose, cellulose-chitosan and cellulose-PMMA

Electrospun cellulose and cellulose composite fibers were fabricated usinga syringe pump, a polypropylene spinning stand, and a spinneret (MECC, Ogori,Fukuoka, Japan). The cellulose and composite solutions were filled in a 10mL syringethat was connected to the spinneret using Teflon tubing. The spinneret was fittedwith a stainless steel needle of internal diameter of 0.94 mm. For electrospinningcellulose fibers, the distance between the needle tip, mounted on the spinningstand, and the aluminum collector electrode was fixed at 9 cm. The flow rate andapplied voltage were optimized to obtain good electrospinnability, with typicalvalues of 90e100 mL/min and 12e15 kV, respectively. Electrospun fibers werecollected into a coagulation distilled water bath to remove [EMIM][OAc] and tosolidify the fibers. The fibers form an entangled web (fiber mat) in the coagulationbath, and were recovered and washed with distilled water twice and dried undervacuum to remove residual solvent. The resulting fiber mat was freeze-dried to forma loose structure shaped like a cotton ball. Electrospun cellulose-chitosan fiberswere prepared by electrospinning at 20 kV, with 95 mL/min flow rate and fixeddistance of 8 cm between needle tip and collecting electrode. Cellulose-PMMA fiberswere electrospun at 19e21 kV, with 50 mL/min flow rate and 8.5 cm distance.

2.4. Lst conjugation with cellulose, cellulose-chitosan and cellulose-PMMA

The electrospun cellulose fibers were oxidized by incubation with 0.5% (w/v)sodium periodate prepared in 100 mM sodium phosphate solution at pH 5.7 withshaking at 180 rpm at room temperature (RT) for predetermined periods of time, 0.5,3, 6.5 and 9.0 h. The reaction was stopped by adding 1:1 water:ethylene glycolmixture followed by washing with water to remove excessive NaIO4. The aldehydegroups containing oxidized cellulose fibers were incubatedwith 2mL of 1mg/mL Lst

J. Miao et al. / Biomaterials 32 (2011) 9557e9567 9559

(AMBI Products LLC.) prepared in Dulbecco’s phosphate buffered saline (PBS, 10mM,pH 7.4) at 4 �Cwith shaking at 160e180 rpm. The conjugationwas stopped after 24 hand the oxidized cellulose-Lst composite fibers were washed twice with water andtwice with PBS to remove non-specifically bound Lst. The cellulose-Lst compositefibers were further reduced for 2 h at RT with 1.5% sodium cyanoborohydride in PBS.

Cellulose-chitosan composite fibers were functionalized with aldehyde groupsby reacting overnight at 45 �C with excess 3.1% glutaraldehyde in 100 mM sodiumphosphate solution at pH 8. The resulting aldehyde-functionalized cellulose-chito-san composite fibers were washed thoroughly with water to remove residualglutaraldehyde and then incubated in 2 mL of 1 mg/mL Lst solution prepared in PBS.The conjugation process was similar to that used for conjugation of oxidizedcellulose fibers with Lst.

Cellulose-PMMA composite fibers were hydrolyzed with 1 M NaOH at RT for24 h and neutralized with diluted HCl to afford carboxylic acid groups on thecomposite fiber surface. Lst was conjugated to the hydrolyzed cellulose-PMMAcomposite fiber using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-chloride (EDC, 100 mM) and N-Hydroxysuccinimide (NHS, 50 mM) (PierceBiotechnology, Rockford, IL) in sodium phosphate solution at pH 5.7 at 4 �C and160 rpm for overnight. The resulting conjugated cellulose-PMMA-Lst compositefibers were washed with water and PBS.

2.5. Thermal behavior and mechanical characterization

Thermogravimetric analysis (TGA) was performed using a computer-controlledTA Instruments TGAQ50 (New Castle, Delaware, USA). The temperaturewas rampedup at 20 �C per min to 700 �C with the furnace open to allow airflow along withnitrogen gas purge. Tensile strength measurements were performed using an Ins-tron Materials Testing Machine (Model 5543, Norwood, MA) equipped with a 10 Nstatic load cell and hydraulic grips (Instron 2712-001). Specimens were tested at0.25 mm/min tension speed, with a 5 mm gauge length. Both load and grip-to-gripdistances were measured. Tensile strength was calculated by dividing the maximumload by the initial cross-sectional area of the sample.

2.6. Surface morphology characterization

Field emission-scanning electron microscopy (FE-SEM) was performed to studythe surface morphology of fibers with a JEOL JSM-6335 FE-SEM (Tachikawa, Tokyo,Japan) equipped with a secondary electron detector at an accelerating voltage of10 kV and at a working distance of 15 mm. Prior to performing the FE-SEM analysis,fibers were coated with gold by sputtering to form a conductive layer.

2.7. X-ray photoelectron spectroscopy (XPS)

XPS experiments were operated in an ultra high vacuum chamber usinga hemispherical electron energy analyzer (HA100, VSW Ltd., UK) and a mono-chromatized Al X-ray source. The spectra were collected at a take-off angle of 45� .Survey spectra were acquired with pass energy of 117.4 eV. The binding energy scalewas calibrated using the aliphatic component of the C 1s peak as 285.0 eV.

2.8. Time-of-flight secondary ion mass spectrometry (Tof-SIMS)

Tof-SIMS instrument was equipped with an Au primary ion cluster sourceoperating at 30 keV (Physical Electronics, Eden Prairie, MN). High mass resolutionspectra were obtained by the use of bunched primary ions. The analysis area wasw300 � 300 mm. Positive and negative secondary ions spectra were acquired andmass calibration of the spectra was based on CHþ

3 , C2Hþ3 , and C3H

þ5 cations, and CN�

and CNO� anions.

2.9. FTIR spectroscopy

FTIR spectroscopy was conducted using a PerkineElmer Spectrum One FourierTransform Infrared Spectrometer. The solid samples were first mixed with KBr andground to a fine powder by using a mortar and pestle, and then pressed into pelletsunder compression pressure of 2000 kgf/cm2 for 30 s. Sample spectrawere obtainedusing an average 32 scans over the range between 800 and 4000 cm�1 witha spectral resolution of 2 cm�1.

2.10. Porosity measurements of cellulose-based fiber mats

The thickness of rectangular shaped fibers mats (0.64 � 0.5 cm) was measuredby use of a micrometer, and the apparent density and porosity were estimated usingEqs. (1) and (2) [46].

rapp ¼ Mfm�Lf���Wf

���Tf� (1)

εfm ¼�1� rapp

rB

�� 100% (2)

Where rapp and rB are apparent and bulk densities, respectively, εfm is porosity, andLf, Wf, Tf and Mfm are the length, width, thickness and mass of the fiber mat,respectively. rB of the cellulose fiber mat was determined by calculating the densityof mixture solvents, chloroform and ethanol, in which the cellulose-based fiber matwas freely suspendedwithout settling at the bottom of the vial or floating on the top.rB was w1.43 g/cm3.

2.11. Processing of the fibers to generate self-supporting mats

Cellulose-based fiber mats were prepared by filtering the non-functionalizedand Lst-conjugated cellulose fibers onto a 0.2 mm polycarbonate membrane (Milli-pore, Billerica, MA). This was done for two reasons e to avoid the release of indi-vidual fibers and the resulting activity in triton and DI water wash solutions, and tomimic the antiseptic gauges that are used in wound healing applications. Thecellulose mats obtained were freeze-dried. The mats were washed with 1% (w/v)triton solution followed by incubation in water for 3 h at RT to remove the loosely-bound enzyme. The wash solution, thus obtained, was tested against S. aureus tocheck for any release of Lst or Lst-cellulose fibers.

2.12. Preparation of S. aureus suspension

Microbial culture of S. aureus (ATCC 33807, ATCC, Manassas, VA) was grown at37 �C for 16 h at 200 rpm in nutrient broth (3 g/L beef extract, 5 g/L peptone) (Difco,Detroit, MI) prepared in DI water. A 100 mL aliquot from this culture was subse-quently added to 4mL of fresh nutrient broth, whichwas incubated for 6 h under thesame conditions. From this growing culture, 1 mL was centrifuged at 10,000 rpm for5 min at RT to obtain a pellet, which was then washed twice with PBS solution toremove excess medium. Bacteria were then reconstituted in PBS. The optical density(OD) of the microbial suspensionwas measured at 540 nm (w109 CFU/mL/A.U. [47])to obtain an approximate measure of cell density in terms of colony forming units(CFU)/mL. The antimicrobial activity of Lst-functionalized and control cellulosefibers was determined by using a diluted suspension containing w106 CFU/mL.

2.13. Determination of antimicrobial activity

Antimicrobial activity of cellulose fiber mats was determined by incubating thefiber mats in 5 mL of a microbial suspension containing 106 CFU/mL S. aureus at RTand 60 rpm for 3 h. Colony counts, and hence the number of live cells, weredetermined by using a plating assay. Specifically, aliquots were removed from theindividual sample suspensions at the end of incubation, followed by plating of themicrobial suspensions onto nutrient agar and incubation at 37 �C for 18 h. To checkfor the possible release of Lst in the solution, an Lst-functionalized cellulose fibermat was immersed in 5 mL PBS (10 mM, pH 7.4, Dulbecco’s Phosphate BufferedSaline, Sigma) and was shaken at 60 rpm and RT for 3 h. The cellulose fiber mat wasthen removed from the wash solution, and microbes were added to the washsolution so as to attain a final cell density of 106 CFU/mL. The resulting microbialsuspension was then shaken on an orbital shaker at 60 rpm for 3 h. The colonycounts, obtained from plating assay, were used to determine antimicrobial activityeby comparing colony counts in the case of Lst-functionalized cellulose fiber mats tothose obtained for the control cellulose fiber mats.

2.14. Keratinocyte cell culture

Keratinocytes (HaCaT, a gift from Prof Karande’s laboratory at Rensselaer Poly-technic Institute, Troy, NY) were maintained in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) Peni-cillin/Streptomycin mixture at 37 �C and in 5% CO2. The HaCaT cells, when attained70e80% confluency, were used for testing the toxicity of cellulose fiber mats and inpreparing keratinocyte-based skin-like model for testing in vitro antistaphylococcalactivity of the Lst-based cellulose fiber mats or bandage system. Specifically, theHaCaT cells adhered to the culture flask were dissociated by using 0.25% trypsin-containing media (Difco) for 5e10 min at 37 �C. The resulting trypsin-containingcell suspension was neutralized by adding growth media and centrifuged toobtain a pellet that was then dispersed in fresh media. For toxicity and in vitro skinmodel studies, cell suspensions at 3 � 105 cells/mL were prepared in completeDMEM media.

2.15. Cytotoxicity studies

For cytotoxicity studies, monolayer cultures of HaCaT cells were prepared byharvesting the cells in 24-well plates at a seeding density of 3 � 105 cells/well (thenumber of cells required to cover entire surface of a well), followed by incubation at37 �C and in 5% CO2 for 24 h to allow attachment of cells. Formation of monolayerculture was confirmed by observing individual wells under the optical microscope.Subsequently, the exhausted media was removed and was replaced by either 100 mLPBS (in case of the control sample and the mats) or protein solutions. The aqueousprotein samples included bovine serum albumin (BSA), denatured Lst (prepared byheating at 95 �C for 20min) and Lst prepared in PBS,with final concentrations of 2mg/mL. For cytotoxicity studies, 100 mL of protein solution was added to the wells

J. Miao et al. / Biomaterials 32 (2011) 9557e95679560

containing HaCaT monolayer cultures. In the case of solid samples, control oxidizedcellulose, BSA-functionalized cellulose and Lst-functionalized cellulose mats weresoaked in 100 mL PBS dispensed on top of the monolayer cultures. To maintain thegrowing environment for HaCaTcells, 0.9mL of fresh growthmediawas added to eachwell. Lst, cellulose mat, and Lst-functionalized cellulose mat, were incubated with themonolayer cultures in growth media at 37 �C and in 5% CO2 for 24 h, to assess thecytotoxicity against HaCaT cells. Cell-free media samples (50 mL) were removedwithout disturbing the adhered cells after 24 h. This was done to monitor the releaseof lactate dehydrogenase (LDH) from damaged cells, as a measure of the cytotoxiceffects of protein solutions and cellulose fiber mats. The aliquots collected from theexperimental samples were mixed with LDH assay reagents (containing lactate,NADþ, diaphorase, 2-p-(iodophenyl) -3-(p-nitrophenyl)-5-phenyltetrazolium chlo-ride (INT)). LDH catalyzes the dehydrogenation of lactate to pyruvate and NADH onaddition of NADþ. The NADH generated is then used by diaphorase to convert INT intoa formazan product, which is detected by measuring the absorbance at 490 nm. Toaccount for the maximum possible LDH release, PBS-treated cells were lysed byadding Triton� X-100 (final concentration ¼ 1%) or by subjecting the cells toa freezeethaw cycle (by incubating at�80 �C for 2 h, followed by thawing at 37 �C for30 min). The cytotoxicity of Lst-, BSA- and cellulose-based formulations was deter-mined by comparing the LDH release obtained from the treated cells with themaximum LDH release obtained after lysing PBS-treated cells.

2.16. Antimicrobial tests using skin model

A keratinocytes-based skin model was used to mimic a skin scenario wherehuman skin cells are exposed to bacteria. Cells were added to 24-well plate ata seeding density of 3 � 105 cells/well and incubated for 24 h at 37 �C and in 5% CO2.The cell-freemediawas removed and the cells were gently washed three-times withsterile PBS to remove residual antibiotics from the culture. The cells were imagedusing an optical microscope to check for confluency of the HaCaTcell monolayer thatserved as a skin model. S. aureus suspension (100 mL of 107 CFU/mL) was added.Subsequently, the cellulose fiber mats (BSA-functionalized and Lst-functionalized)were placed on top of the S. aureus exposed HaCaT cell layer. The samples, con-sisting of S. aureus-infected HaCaT cells and cellulose preparations, were incubatedfor 3 h at RT. At the end of 3 h incubation, 0.9 mL of sterile PBS was added to eachwell and the 24-well plate was mixed thoroughly. Cells were plated on growth agarand incubated at 37 �C for 16 h to determine the viability of cells in the suspension.The viability of cells adhering to the cellulose mats was determined by transferringthe fiber mats into 1 mL sterile PBS, followed by sonication to re-disperse theadherent cells into PBS and plating of the cells on agar plates. The fiber mats werefirst air-dried and then placed onto an agar plate for 2 min to allow transfer of anyresidual cells and to ensure the complete removal of cells from cellulose fiber matsafter sonication.

3. Results and discussion

3.1. Electrospinning of cellulose, cellulose-PMMA, and cellulose-chitosan

Cellulose solutions in [EMIM][OAc] were electrospun intoa coagulation bath at various solution concentrations, voltages,feeding rates and spinneret-grounded electrode distances. Theseparameters were optimized to obtain continuous jets resulting ingood spinnability. Three concentrations of cellulose solution, 1.5%,1.75% and 2% (w/w) were used. The fibers electrospun froma 1.5% (w/w) cellulose solution afforded a very low yield of fibersdue to formation of droplets during electrospinning. A 2% (w/w)cellulose solution led to thicker electrospun fibers and a 1.75% (w/w) cellulose solution afforded thinner fibers and these wereselected for further experiments. The flow rate was kept below100 mL/min to ensure the formation of relatively thin fibers. Thevoltage of 12e13 kV and distance of 9 cm were also critical formaintaining a continuous jet and higher fiber yields. Fibers formedin the coagulation water bath were collected every 10e15 min andtransferred into 10 mL distilled water to avoid fusion of cellulosefibers due to residual RTIL. Dry fibers were obtained by freezedrying into a loose cotton ball or vacuum drying onto a cover glass.For functionalization of cellulose by chemical oxidation, the elec-trospun cellulose fibers were collected every 10 min to ensure thatsimilar amounts of cellulose fibers were present in each batch. Theresulting wet cellulose fibers were directly used in the chemicalmodification step.

Electrospinning of chitosan-cellulose [EMIM][OAc] solutionneeded further optimization to obtain a continuous jet, as chitosanincreased the solution viscosity, thereby making it more difficult toelectrospin. Dimethyl sulfoxide (DMSO, 10%) was added as a cosol-vent into the cellulose-chitosan [EMIM][OAc] solution to reduce theviscosity, while maintaining the same final concentration requiredfor sufficient chain entanglement for fiber formation [48]. Theaddition of DMSO facilitated the formation of a continuous jet oftwo-component composite fibers. The spinning conditions weresimilar to those for the cellulose fiber, except a higher voltage wasrequired for stable electrospinning. The thermal stability of thecomposite fibers was characterized by TGA (Fig. S1a). The cellulose-chitosan fibers showed a lower decomposition temperature (lowerby 30e50 �C at weight loss of 1%) than that of the individualcomponents. This may result from disruption of perfect crystallinestructure of cellulose and chitosan in the formation of the two-component composite.

The electrospinning of cellulose-PMMA solution was not stabledue to a rapid phase separation process as a result of thermody-namic instability of hydrophobic PMMA in the highly polar RTIL.DMF was used as an additive to ensure the solubility of PMMA andreduce the viscosity. Nonetheless, occasional discontinuity ofspinning still occurred and beads were formed in the fibers. TritonX-100 surfactant was used as a second additive to prevent the jetdiscontinuity and bead formation.

3.2. Characterization of fiber morphology

The fiber morphologies were characterized using FE-SEM. Theelectrospun nonwoven cellulose, cellulose-chitosan and cellulose-PMMA fibers showed smooth surface and cylindrical fiber struc-ture (left panel of Fig. 1). The average pore size of cellulose fiber matis much larger than 1 mm, and the pore size distribution wasnarrow. The pore size of the cellulose-chitosan fibers was muchsmaller than that of the cellulose fibers. The appearance of fusedfibers may be caused by chitosan gel formation during the coagu-lation process. A larger pore size and a wider distribution wereobserved for the cellulose-PMMA fibers, possibly due to an unstablespinning process caused by the inhomogeneous electrospinningsolution. The diameter distributions of oxidized cellulose fiber,cellulose-chitosan and cellulose-PMMA are shown in Fig. 1 (rightpanel). Oxidized cellulose fibers had an average diameter of ca.450 nm with a standard deviation of 126 nm. The cellulose-PMMAand cellulose-chitosan had smaller average diameters(304e308 nm), likely attributable to the lower solution viscositydue to the addition of DMF or DMSO. The broad distributions ofcellulose-chitosan and cellulose-PMMA fibers revealed by largerstandard deviations of 156 nm and 171 nm, respectively, may becaused by phase separation.

3.3. Functionalization of cellulose, cellulose-chitosan and cellulose-PMMA fibers

The cellulose fibers were activated by oxidation with 0.5%sodium periodate (see Materials and Methods) [49,50]. FTIR spec-troscopy was performed to analyze the amount of C]O (aldehydegroup at w1730 cm�1) produced to test the functionality of cellu-lose fiber (Fig. 2). For oxidation times of 0.5 and 3.5 h, there is noobvious shoulder on the left of OeH bending peak at 1635 cm�1 dueto absorption of moisture. At 6 h, a band gradually appeared atw1730 cm�1 suggesting formation of aldehyde groups. An optimaloxidation time of 9 h was selected to maintain the mechanicalstrength, as a prolonged oxidation reaction dissolved the cellulose.The resulting oxidized cellulose fibers were conjugated with Lstwithout further drying or processing.

Fig. 1. SEM images (left panel) for oxidized cellulose fibers, cellulose-chitosan fibers and cellulose-PMMA fibers from top to bottom, and size distribution (right panel) for cor-responding SEM images.

J. Miao et al. / Biomaterials 32 (2011) 9557e9567 9561

Electrospun cellulose-chitosan fibers were functionalized byreacting with glutaraldehyde to afford an aldehyde group-containing fiber surface. The degree of reaction was monitored byFTIR (Fig. S2). After cross-linking, an additional band as shown bythe arrow appeared at 1712 cm�1, which is very close to theabsorption maximum for C]O in glutaraldehyde (1714 cm�1),confirming that the cellulose-chitosan composite fibers weresuccessfully functionalized (Fig. 2).

Cellulose-PMMA fibers were functionalized by hydrolysis ofPMMA in NaOH solution (see Materials and Methods). The hydro-lysis was alsomonitored by FTIR. As the reaction proceeded, a broadband arose at w1704 cm�1, associated with the C]O stretchingfrom the free carboxylic acid (Fig. S3). Another indication of

progress of hydrolysis is the reduced intensity of peak at 841 cm�1,characteristic of a methyl ester.

3.4. Surface characterization of Lst on the cellulose fibers

Because of considerable nitrogen content in Lst (w17.3%, w/w)the loading of Lst onto oxidized cellulose fibers was quantified bydetermining nitrogen content by elemental analysis (3% w/w). It isalso possible to analyze the Lst distribution across the oxidizedcellulose fiber mat by using XPS. This method is not suitable forcharacterization of cellulose-chitosan due to the high nitrogencontent of chitosan. XPS is capable of quantitatively measuring theelemental compositions at a depth of up to 10 nm from the

Fig. 2. FTIR spectra for oxidized cellulose fibers at 0.5, 3.5, 6 and 9 h. Arrow indicatesthe appearance of aldehyde peak at 1730 cm�1.

J. Miao et al. / Biomaterials 32 (2011) 9557e95679562

surface. Strong C1s and O1s peaks were observed in both thecontrol oxidized cellulose fibers and oxidized cellulose-Lst fibers(Fig. 3). An additional N1s peak was observed in the oxidizedcellulose-Lst fibers, confirming the conjugation of Lst. Tof-SIMSwas applied to identify the molecules and their correspondingsecondary ion distribution on the surface of fiber mat with a depthresolution of 1e2 nm. Regions in the control oxidized cellulosefibers that showed characteristic positive ion peaks from cellulose,CH2Oþ at m/z ¼ 30.01 and C4H6O

þ2 at m/z ¼ 86.04 (Fig. 4). In

addition to the two peaks observed for cellulose, other severalpositive ion peaks were observed for oxidized cellulose-Lst fibersthat could be attributed to the individual amino acids from Lst: Gly(CH4Nþ at m/z ¼ 30.04), Ala (C2H6Nþ at m/z ¼ 44), Asn (C3H4NOþ

at m/z ¼ 70), Val (C4H10Nþ at m/z ¼ 72), and Leu (C5H10Nþ at m/

Fig. 3. XPS spectrum for oxidized cellulose fiber and Lst conjugated oxidized cellulose

z ¼ 86.10). These assignments for characteristic positive ions forindividual amino acids are consistent with reported ToF-SIMS dataon amino acids with different substrates [51]. The negative ions,CN� and CNO�, are used for mapping the nitrogen distribution(Fig. 5) as they come from the protein backbone, which is distinctfrom the cellulose substrate [52]. Within an area ofw300 � 300 mm (Fig. 4b), black patches correspond to 0% signal,gray to intermediate signal, and white to 100% signal. The imageswere normalized setting the highest intensity to 100% signal. TheN-distribution from CNO� peak (m/z ¼ 42) in Fig. 5, identified bywhite or gray dots, was uniform indicating that Lst molecules werehomogeneously distributed on the surface of the fiber-basedmembranes.

3.5. Antimicrobial activity of Lst-cellulose fibers

The three cellulose-based and Lst-functionalized fiber prepara-tions were tested for their antimicrobial activity against S. aureus.To confirm contact-mediated killing, fiber-basedmats werewashedwith water and the resultant wash solutions were tested against106 CFU/ml S. aureus suspension. In the case of cellulose-chitosan-Lst fibers, where the enzyme was covalently bound to fibersthrough glutaraldehyde cross-linking, the bioconjugation wasstable as reflected by the insignificant antimicrobial activity in thewash solution (Fig. 6a). The control cellulose-chitosan fibersshowed w20% bacterial killing after a 3 h incubation. This resultwas not unexpected, as chitosan is known to be antimicrobial dueto its polycationic nature [53]. Nonetheless, functionalization ofthese moderately antimicrobial cellulose-chitosan fibers with Lstresulted in >95% bacterial killing. The relatively lower antimicro-bial activity compared to Lst-functionalized nanotube/paint films[24] could be due to a high degree of cross-linking leading toreduced mobility of the attached enzyme.

Cellulose-PMMA-Lst fiber-based mats prepared through conju-gation carried out by carbodiimide chemistry, showed some level ofenzyme leaching when fibers were washed with water. The factthat there was activity in the wash solution suggests that some ofthe enzyme had been physically adsorbed onto hydrophobicregions in the cellulose-PMMA hybrid fibers. The cellulose-PMMA-Lst fiber preparations were highly active, however, leading tocomplete neutralization of microbial challenge (Fig. 6b).

fibers; the circle indicates the appearance of nitrogen peak exclusively from Lst.

Fig. 4. Mass spectra from Tof-SIMS for control oxidized cellulose fibers and Lst conjugated oxidized cellulose fibers.

J. Miao et al. / Biomaterials 32 (2011) 9557e9567 9563

Lst was attached to periodate-treated cellulose fibers via forma-tion of a reversible Schiff base, which is reduced to form a stablecovalent bond. As a result of such stable bioconjugation, no antimi-crobial activity was observed in the wash solution (Fig. 6c). Theoxidized cellulose-Lst fiber mat showed complete neutralization ofbacterial challenge following a 3 h incubation (Fig. 6c). Since thisstable bioconjugation had high antimicrobial efficacy, oxidizedcellulose-Lstfiberswereused throughout the remainderof this study.

3.6. The effect of porosity on cell killing

The porosity of the fiber mats will govern the infiltration ofthe bacterial suspension inside the fiber matrix, and hence, the

Fig. 5. Nitrogen mapping for Lst conjugated oxidized cellulose fiber (a) and control oxidizeintensity and black no nitrogen signal. The scale bar is 100 mm.

accessibility of Lst molecules buried inside the fiber matrix.Specifically, at low matrix porosity, bacterial cells will only comein contact with the surface-exposed enzyme, thereby limitingantimicrobial activity to the exposed surface of the fiber mat.High porosity will allow cell penetration into the 3-D fiber mat,which will increase the possibility of bacterial cells contactingthe Lst-functionalized fibers, and thus, lead to an increase inbactericidal activity [54]. The desired porosity of the fiber matfor cell infiltration has been reported to be in the range of60e90% [46]. In the current work, the porosity for Lst-functionalized oxidized cellulose fiber mat was calculated tobe 78e84%, which is sufficient for cell infiltration and thereforefor bacterial killing.

d cellulose fiber (b). The white dots indicate high intensity of N signal, gray dots low

Fig. 6. Antistaphylococcal activity of various Lst-containing cellulose fiber formulations and corresponding wash solutions: (a) cellulose-chitosan and cellulose-chitosan - Lst fibers,(b) cellulose-PMMA and cellulose-PMMA-Lst fibers, and (c) oxidized cellulose and oxidized cellulose-Lst fibers. In each case, the activity for control and Lst-based cellulose fibers isshown in gray and white bars, respectively. (*) indicates that no colonies were observed in the corresponding samples.

J. Miao et al. / Biomaterials 32 (2011) 9557e95679564

3.7. Biocompatibility of cellulose-Lst fiber mats

In case of an external infection, either through wounds or burns,human skin is the first body organ that comes in contact withpathogenic bacteria. Human skin is composed of the epidermis anddermis, with the epidermis constituting the exterior layer andpredominantly comprised of keratinocytes [55]. Therefore, kerati-nocytes were selected to study the biocompatibility of Lst-basedcellulose mats. Along those lines, monolayer cultures of HaCaTcells were used as the reconstructed epidermis model, for evalu-ating cytotoxicity [56,57].

An LDH release assay was employed to assess the viability ofHaCaT cells following exposure to Lst, cellulose and cellulose-Lstfiber mats. Solutions of BSA and denatured Lst, prepared in PBS,served as aqueous control samples. BSA-functionalized cellulosefiber mats were used as a control for experiments involving

Fig. 7. LDH release assay to test cytotoxicity of (a) protein solutions or (b) protein based cellubased preparations normalized to the freeze-thawed cells (positive control) and cell-free m

exposure of HaCaT monolayer cultures to cellulose fiber mats. Asa positive control and to account for the total LDH associatedwith target cells, PBS-treated cells were lysed by subjectingthem to one freezeethaw cycle. Cell-free media collected fromPBS-treated monolayer culture served as the negative control.The cytotoxicity of different protein and cellulose formulationswas determined by comparing the LDH activity observed in thecase of cell-free samples, collected from each treatment, to theLDH activity from completely lysed cells. As shown in Fig. 7a,protein-containing preparations showed same level of cytotox-icity as in the case of PBS-treatments, suggesting insignificantrole of protein samples in the observed cytotoxicity. Addition-ally, BSA- and Lst-functionalized cellulose mats showed<5% cytotoxicity (Fig. 7b). Thus, these results indicate thatcellulose-based anti-infective preparations are non-cytotoxic toHaCaT cells.

lose mats against keratinocytes. The plots show % cytotoxicity of protein- and cellulose-edia from PBS-treated cells (negative control).

Fig. 8. (a) A skin model to assess the anti-bacterial activity of Lst-functionalized cellulose mats e a three layer model consisting of keratinocytes, S. aureus suspension, and cellulosemats; (b) % Viability of S. aureus cells in free solution when treated with different formulations in the presence of keratinocytes; and (c) % Viable S. aureus cells present on differentcellulose-based mats. (*) no colonies were observed in these samples, suggesting complete sterilization.

J. Miao et al. / Biomaterials 32 (2011) 9557e9567 9565

3.8. Antistaphylococcal activity in a skin-like model

We selected a keratinocyte-based in vitro system to mimic skinirritation in this study [56]. A monolayer culture of HaCaT cells(grown for 24 h in a 24-well plate) was incubated with S. aureussuspension and Lst-functionalized cellulose fiber mats tomimic theinfection treatment scenario, where the skin surface becomesexposed to bacterial cells and a bandage preparation (Fig. 8a). Theanti-infective activity of oxidized cellulose-Lst preparation wasmeasured by checking the viability of S. aureus cells in freesuspension as well as on cellulose preparations. Lst-containingcellulose preparation resulted in complete neutralization ofS. aureus compared to control oxidized cellulose fibers (non-func-tionalized and BSA-containing) (Fig. 8b). Additionally, the cellulose-bound cells were found to be completely damaged in the case of Lsttreatment in contrast to those bound to control samples (Fig. 8c).Thus, we demonstrated the antistaphylococcal activity of Lst-

functionalized cellulose preparations when treated againstS. aureus infecting a monolayer HaCaT culture.

4. Conclusions

Three different cellulose fiber preparations were obtained byelectrospinning solutions of cellulose, cellulose-chitosan, andcellulose-PMMAmade in [EMIM][OAc]. These electrospun cellulosefibers were chemically treated to obtain enzyme-reactive func-tionalities through oxidation, cross-linking, and hydrolysis, and theefficacy of Lst conjugationwas assessed. FE-SEM characterization ofthe fibers demonstrated their sub-micron diameter and porosity.Tensile strength testing showed a reasonable (6.0 � 0.5 MPa)mechanical strength for oxidized cellulose fibermat. Three differentLst-based fiber conjugates were prepared by immobilizing Lstthrough Schiff base formation and carbodiimide chemistry. TheseLst-cellulose biohybrids were processed to form self-standing fiber

J. Miao et al. / Biomaterials 32 (2011) 9557e95679566

mats and tested for their antimicrobial properties against S. aureus.The optimal anti-infective formulation was based on Lst bound tooxidized cellulose fibers. Tof-SIMS revealed the uniform distribu-tion of Lst on the oxidized cellulose fibers, based on the mapping ofsecondary negative ions CNO�, which are specifically associatedwith the protein backbone. Secondary positive ions were alsopresent from individual amino acids (i.e., Gly, Ala, Asn, Val and Leu)of the Lst molecules. Antimicrobial assays performed on the Lst-based oxidized cellulose fiber preparations suggested completeneutralization of exposed S. aureus cells; hence, in vitro cell culturetechniques were employed to test the biocompatibility of thesepreparations against keratinocytes (HaCaT cell line). LDH releaseassays revealed minimal cytotoxicity of control and Lst-basedoxidized cellulose preparations against HaCaT cells. Additionally,the antistaphylococcal effect of these preparations was demon-strated in a skin-like model consisting of monolayer culture ofHaCaT cells. Our findings suggest that these anti-infective and non-cytotoxic Lst-based oxidized cellulose fiber preparations potentiallycan be used in wound healing applications.

Acknowledgments

This work was supported by the U.S. National Science Founda-tion funded Rensselaer Nanotechnology Center as well as ChissoCorporation. We thank Prof. Pankaj Karande (Rensselaer Poly-technic Institute) for providing HaCaT keratinocyte cells, used in thein vitro epidermis model studies. Gregory Fisher (Physical Elec-tronics, Inc.) is acknowledged for assistance with Tof-SIMS analysis.

Appendix. Supplementary material

Supplementary material associated with this article can befound, in the online version, at doi:10.1016/j.biomaterials.2011.08.080.

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