Antimicrobial and antioxidant surface modification toward a new silk-fibroin (SF)-l-Cysteine material for skin disease management

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ContentslistsavailableatScienceDirect

Applied

Surface

Science

j o u r n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / a p s u s c

Antimicrobial

and

antioxidant

surface

modiﬁcation

toward

a

new

silk-ﬁbroin

(SF)-

l-Cysteine

material

for

skin

disease

management

Frederico

Nogueira

a,b

_,

_Luíza

_Granadeiro

a

_,

_Claudia

_Mouro

b

_,

_Isabel

_C.

_Gouveia

b,∗

a_CICS-UBI_–_Health_Sciences_Research_Centre,_University_of_Beira_Interior,_Portugal

b_FibEnTech_–_Fiber_Materials_and_{Environmental}_{Technologies,}_University_of_Beira_Interior,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7September2015

Receivedinrevisedform1December2015 Accepted21December2015

Availableonline23December2015 Keywords:

l-Cysteine(l-Cys) Antimicrobialapplications Medicaltextiles Atopicdermatitis(AD) Staphylococcusaureus

a

b

s

t

r

a

c

t

Anoveldressingmaterial–silkﬁbroinfabric(SF)-l-Cysteine(l-Cys)–isheredevelopedtobeusedas

standardtreatmentforatopicdermatitis(AD),whichcombinescomfort,thermic,andtensilestrength

propertiesofsilkmaterialswithantioxidantandantimicrobialeffectsofl-Cys.Acareful

understand-ingaboutthelinkingstrategiesisneededinordernottocompromisethebioavailabilityofl-Cysand

deplenishitsbioactivity.Durabilitywasalsoaddressedthroughwashingcyclesandcomparedwith

hos-pitalrequirements,accordingtointernationalStandardENISO105-C06:2010.Thepresentresearchalso

analyzetheinteractionsbetweenStaphylococcusaureusandSF-l-CysundersimulatingconditionsofAD

anddemonstratedtheeffectivenessofadoublecovalentgrafting,withtheimportanceofSFtyrosine

(Tyr)covalentlinkagewithl-Cys(SF-g-l-Cys/Tyr-g-l-Cys)evenafterseveralwashingcycles,twentyﬁve,

whereasforadisposableapplicationasinglecovalentmechanismofgraftingl-Cysprovedtobe

sufﬁ-cient(SF-g-l-Cys).Resultsshowedeffectiveantimicrobialactivitiesexhibitinghigherinhibitionratiosof

98.65%forSF-g-l-Cysafter5washingcycles,whereas97.55%forSF-g-l-Cys/Tyr-g-l-Cysafter25washing

cycles,bothatpH9.5graftingstrategy.Furthermore,itisalsoreportedanon-protumoraleffectofl-Cys.A

newadvanceishereinachievedattheworldofmedicalantimicrobialtextilestailoredtoaddresswound

moistureenvironmentandexudateself-cleaning,whichmayopennovelapplicationsascomplementary

therapyforADdisease.

1. Introduction

Silk,producedbysilkwormBombyxmorii,consistsofﬁbroinand

sericin.Fibroin,whichisthestructuralproteinofsilkﬁbers,comes

outfromthesilkwormglandintheformofﬁlamentsenclosedina

water-solublesericingluelikecoating[1].Silkﬁbroin(SF)is

com-posedofheavy (350kDa)and lightchain (25kDa)polypeptides

linkedbyadisulﬁdebond[2,3].Itsstructureismainlycomposed

bytheaminoacidsglycine(46%),alanine(29%),andserine(12%),

whichareaminoacidshavingsmallsidechains.Also,SFpresents

tyrosine(4.8%),whichhasanaromaticsidechain[4].Theyform

ﬂexibleandstretchingresistantsecondarystructurebeta-sheets,

whichrendersilkitscharacteristicmechanicalstrength[5–7].Silk

isnon-toxic,non-immunogenic, biocompatible,andhydrophilic.

AlthoughitisstatedthattheitchingofADisexacerbatedbyspiky

∗ Correspondingauthorat:FibEnTechR&D–FiberMaterialsandEnvironmental Technologies,UniversityofBeiraInterior,RuaMarquêsd’ÁvilaeBolama,6201-001 Covilhã,Portugal.

E-mailaddress:igouveia@ubi.pt(I.C.Gouveia).

ﬁbersofwoolandbythewildtextureofcotton[8,9],silk,onthe

otherhand,withitsextremelysmoothanditscomfortable

prop-ertiesreducestheseeffectsand thereforehasbeenincreasingly

researchedforapplicationsrelatedtoatopicdermatitis(AD).

Fur-thermore,Sugiharaetal.[10]postulatedthatwoundstreatedwith

asilkdressinghealed7daysfaster,bypromotingtheenhanced

synthesisofcollagenandepithelialization[10].

AD,alsoknownaseczema,isachronicandrecurrent

inﬂamma-torypruriticskindisorderwhichcauseslicheniﬁcation,papules,

andexcoriations.ADhasbeenincreasingoverthepastdecades,

reachingprevalenceof about 20% in children and 3% in adults

worldwide [11]. A combination of genetic, environmental, and

immunologicalfactorsmaybethemaincausesforthe

develop-mentofAD,whoseconsequencescanbepsychologicallytraumatic,

leadingtoemotionalstressanddecreasingsubstantiallythequality

oflife.ADischaracterizedbyacombinationofT-helpertype(Th)

2immuneactivation,skininfection/colonizationby

Staphylococ-cusaureus,andskinbarrierdysfunction.ADprevalencerisemay

beexplainedbytoday’slifestyleand nutrition[11].Thecleaner

environmentinupbringingaswellasantibioticsabusingduring

earlychildhoodhasinducedmoreoftenTh2immuneresponses,

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aretriggeredbyS.aureusproducingtoxinsordirectinvasionand

destructionoftissues.Notwithstandingthatclinicalmanifestations

ofdiseaserelyalmostexclusivelyontoxinactivity,inminor

situa-tionsitcanalsobeduetobacterialproliferationthatformsabscess

andtissuedestruction.Italsopresentsresistanceagainstthe

cur-rentlyusedantimicrobials[18].

Inthepresentworkwehavechosenl-Cysteine(l-Cys),which

isa␣-aminoacidwithathiolgroupattheendofitsvariableside

group,whichisnucleophilicandhasawidespectrumofbioactivity

[19],relyingitsactivityonmetabolicdisturbanceandmembrane

depolarization[20].Ittargetsthebacterialmembraneandcauses

agreatdecreaseinenzymaticactivityandbacterialmetabolism.

Owingtoitsabilitytoformconjugateswithfreeradicalsortrace

elements[21],l-Cysmayalsorenderourﬁnalapplication

antioxi-dantproperties.

Thisresearchworkconsistedonthedevelopmentoftwo

pro-cesses that allowed the covalent grafting of _l-Cys onto SF: (i)

functionalizationofSFcarboxylates(degradedamidegroups)and

cross-linkingwiththeaminecompoundsofl-Cys;(ii)

function-alizationofSFTyrwithl-Cys,withtheformationof Schiffbase

intermediatesthatwerechemicallystabilizedbyreductive

ami-nation.

Thisallowedtodrawimportantconclusionsregardingthebest

way to pursuit for a disposable application, as well as when

designed for a multi-patient withlaundry-resistant capabilities

(after25washingcycles).

2. Materialsandmethods

2.1. Materials

SilkwasobtainedfromthecocoonsoflarvaeofthesilkwormB.

morii(Brazil).Silkwasknittedatarectilinearneedleloom with

7Picks perinch,witha Jersey knitstructure anda linear

den-sityof21.68numbermetric(Nm).Itssericinwasremovedwith

dichloromethaneinasoxhletapparatus.Silkﬁbroin(SF)wasthen

functionalized in a _l-Cysteine (_l-Cys) (Sigma–Aldrich) solution

(ddH2O)atpH’s5.5and9.5.Covalentbondsattyrosine(Tyr)were

performedwithNaCNBH3dissolvedinNaOH:HClbuffers(Panreac).

S.aureus(ATCC6538)strainwasgrownonNutrientAgar(NA)and

NutrientBroth(NB)(Panreac).

2.2. Methods

2.2.1. Minimalinhibitoryconcentration(MIC)evaluation

Minimalinhibitoryconcentration(MIC)determinationwas

per-formedwiththemicrodilutionmethodinaccordancewithM07-A6

radicalthatcanacceptanelectronorhydrogenradicaltobecome

astablediamagneticmolecule.DPPH◦ bearsadeeppurplecolor,

butoverthecourseofthereactionbecomescolorless,whenthe

oddelectronofitsnitrogenatomisreducedbyreceivinga

hydro-genatomfromtheantioxidanttestingcompoundl-Cys.TheDPPH◦

assaywasadaptedaccordingtotheproceduredescribedby[22].

Brieﬂy,thestocksolutionwaspreparedbydissolving4mgDPPH

with100mLmethanol.Thepercentage of remainingDPPH◦ for

eachconcentrationofl-Cys wasdeterminedusingtheequation

oftheobtainedcalibrationcurveandabsorbancevaluesmeasured

at517nm,at30mintimepoint:

%DPPH◦REM=[DPPH◦]T/[DPPH◦]T=0×100% (1)

Efﬁcientconcentration(EC50␮g/mL)wasdeﬁnedassubstrate

concentrationtoproduce50%reductionoftheDPPH◦.

2.2.3. Tumoralactivityassay

Thecytotoxicityofl-Cyswasperformed,adaptingthe

proce-duresfromStandardEDINENISO10993-5.Itwasselectedthe

concentrationofl-Cyschosenforthiswork,1%,anditshalf0.5%,on

breastcancercellline(NCI-PBCF-HTB22(ATCC®_HTB-22TM_)).

Incu-bationwasdoneat37◦Cfor48hunderahumidiﬁedatmosphere

containing5%CO2.MTTviabilityassaywasperformed.Solvent,

positiveandnegativecontrolswereincluded.

2.2.4. Silksubstratepreparation

2.2.4.1. Silkmaterial. 20gofSFknittedfabricwasboiledfor4hin

dichloromethaneinasoxhletapparatustoextractresiduallipids,

andthenrinsedthoroughlywithdistilledwater.Itwasthenwashed

according toBS4923: 1991– methods for individualdomestic

washinganddryingforuseintextiletesting,ina4g/Lsolutionof

ECEReferenceDetergent(theformulationofthedetergentisgiven

inBS1006:CO6(ISO105:CO6))for60minat40◦C,rinsed,anddried

afterwardsat30◦Cuntilused,inordertoremoveimpuritiesand

soils.

2.2.4.2. Grafting/functionalizationprocesses. SFknittedfabric

sam-ples werefunctionalized bymeansof anexhaustion processat

90◦C,15rpm,inaMathis-BFA12ﬁnishingmachine.Single

cova-lentstrategy (SF-g-l-Cys):sampleswereimmersedina1%over

theweightoftheﬁber(owf)l-Cyssolution(ddH2O)adjustedto

pH’s5.5and9.5,for3h.Fig.1A.Doublecovalentstrategy(SF-g-

l-Cys/Tyr-g-l-Cys):sampleswereimmersedina1%overtheweight

oftheﬁber(owf)l-Cyssolution(ddH2O)adjustedtopH’s5.5and

9.5,for3h,andwereadded0.3(w/V)NaCNBH3solutionfromthe

beginningofsecondhour.Sampleswererinsedthreetimeswith

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Fig.1.Strategiesfortheactivationofsilkﬁbroin.(A)SF-g-l-Cys;(B)SF-g-l-Cys/Tyr-g-l-Cys.

Firstly,SFknittedfabricsampleswerefunctionalizedonits

car-boxylates(degradedamidegroups)andwerecross-linkedwiththe

aminecompoundspresentinl-Cyssolution(SF-g-l-Cys).Secondly,

SFknittedfabricsampleswerefunctionalizedonitsTyrketone

groups,withtheformationofSchiffbaseintermediatesthatwere

chemicallystabilizedbyreductiveamination,formingultimatelya

covalentbond(SF-g-l-Cys/Tyr-g-l-Cys).

2.2.4.3. Washingcyclesassay–resistancetolaundry.Sampleswere

(i)washedina4g/LsolutionofAATCC1993StandardReference

DetergentWOBfor 5washingcyclesperformedat40◦C during

30mineach, and(ii)washedin a 4g/Lsolutionof AATCC1993

Standard ReferenceDetergent WOB for 25 washing cycles

per-formedat40◦Cduring30mineach.Thesemethodswereadapted

fromtheinternationalstandardEN ISO105-C06:2010.Washing

cyclesweredonepriortoallstudiesinordertoassesstheresistance

towashingofl-CysbondsonSFsurface.

2.2.5. Grafted-silkcharacterization

SFsampleswerecharacterizedusingdifferentsurfaceanalyzing

techniquesagainstappropriatecontrols.

2.2.5.1. Contact angle. Contact angles were measured using an

OCAH-200set-upbyDataphysicsequippedwithahigh-speedvideo

systemwithCCDvideocamerawitharesolutionof752×484.

2.2.5.2. Fouriertransforminfraredspectroscopy(FT-IR).The

chemi-calcompositionofSF,SF-g-l-Cys,andSF-g-l-Cys/Tyr-g-l-Cyswas

analyzedbymeansofFT-IR.Afterthreerinses(nowashingcycles),5

washingcycles,and25washingcyclesFT-IRspectrawereanalyzed.

MeasurementsweredonewithaThermo-Nicoletis10FTIR

spec-trophotometer.Eachsamplewasscanned64times,withaspatial

frequencyresolutionof4cm−1.

2.2.5.3. Exhaustion rates: degree of functionalization.In order to

assesstheefﬁciencyofthefunctionalization,i.e.thequantityof

l-CysthatthesubstrateSFwasabletocrosslink,exhaustionrate

offunctionalizationassayswereperformedforeachcondition.

Ell-man’sreagent,(5,5-dithio-bis-(2-nitrobenzoicacid/DTNB)(Sigma)

produces a yellow color when bonded to free thiol groups in

solution.Afterreactingwithfreethiolgroups(ofl-Cys)thereis

formationofa dissulphydriccompoundand acoloredspecies–

2-nitro-5-thiobenzoicacid(TNB).Thiscolorimetricassaywas

per-formedbymeasuringandcomparingthefunctionalizationsolution

beforeandafterfunctionalization,afterdoingacalibrationwith

knownconcentrationsofl-Cys.Foreachsample250␮Lwasadded

toatesttubecontaining2.5mLofsodiumphosphate(pH8)and

50␮LofEllman’sreagent.Testtubeswerevortexedandwere

incu-batedfor15minutesbeforetheirabsorbancebeingreadat412nm.

2.2.5.4. Tensilestrengthassays. Inordertoevaluatethe

degrada-tioncausedonSFbythefunctionalizationprocesses,severalquality

controlassayswereperformed.Resistancetoruptureassayswere

doneinallsamples,withandwithoutl-Cys,tosubsequent

com-parison.AssayswereperformedonaDynamometer,accordingto

StandardENISO2062,at20±2◦C,under60%relativehumidity.

Sampleswerestrip-cut5cm×50cm,alsostringcut,andplaced

betweendynamometertweezers.Adeﬁnedpre-tensionwassetin

thebeginning,andthetestendedupwithsamplesrupture.

2.2.6. Antimicrobialactivityassessment

TheantimicrobialbehaviorofSFsamplesfunctionalizedwith

l-Cys(-g-l-Cysand-g-l-Cys/Tyr-g-l-Cys)wastestedaccordingto

JapaneseIndustrialStandard JISL1902:2002,speciﬁcfortextile

samples.Gram-positiveS.aureus(ATCC6538)strainwaschosen

onceitplaysanimportantroleinatopicdermatitis(AD).Brieﬂy,

inoculum of S. aureus was preparedwith a ﬁnal concentration

of1–5×106_,_bacteria/mL.₂₀₀_␮L_of_S._aureus_were_inoculated_on

0.4gsquaresamples.Sampleswereincubatedfor24h.

Antimicro-bialactivitywasassessedbyaquantitativemethodtodetermine

thepercentageofbacterialgrowthinhibition(%ofInhibition)at

24hours:

%Inhibition= C−A

C ×100

where C representstheaveragevalueof ColonyForming Units

(CFU-␮g/mL) ofnon-modiﬁedSF,and A representstheaverage

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Imageswereformedthroughsecondaryelectronsusingahigh voltageof20kV.Fiveﬁeldspersamplewereobtainedusinga mag-niﬁcationof20000and75000×.

2.2.8. Transmissionelectronmicroscopy(TEM)

The morphology of adhered S. aureus on SF knitted fabric functionalizedwithl-Cys(-g-l-Cysand-g-l-Cys/Tyr-g-l-Cys)was addressedbyTEM.Bacteriawerestainedwith2%uranylacetatefor 5min,andmountedonameshwiththinbarcoppergridscovered withformvar.Eachsamplewasexaminedat30000×magniﬁcation usingHITACHIHT7700TEMatanacceleratingvoltageof80kV. 2.2.9. Statisticalanalysis

SPSSStatistics21.0softwarewasusedtoperformthestatistical analysisofthedatausingOne-WayANOVA,withTukeyasPostHoc testsforestablishingmultiplecomparisonsbetweensamplesinthis study.Valuesofp<0.05wereconsideredstatisticallysigniﬁcant. 3. Results

3.1. Minimalinhibitoryconcentration(MIC)ofl-Cys

Minimalinhibitory concentration(MIC)of l-Cysteine (l-Cys) againstS.aureuswasfoundtobe5mg/mL.

3.2. Antioxidantactivity

3.2.1. DeterminationofDPPH(2,2-diphenyl-1-picrylydrazyl) radicalscavengingactivity

ThereductioncapabilityoftheDPPH◦radicalwasdetermined bythedecreaseonitsabsorbanceat517nm,inducedbyCysteine. Table1showsthatastheconcentrationofCysteineincreased,the

percentageofremainingDPPH◦diminished.

Thepercentage ofremainingDPPH◦ wasproportional tothe

antioxidantconcentrationofl-Cys.Theconcentrationthatcaused

adecreaseintheinitialDPPH◦concentrationby50%(EC50)was

foundtobe0.029957␮g/mL,whichimpliesthatl-Cysteinehave

antioxidantactivity.

3.3. Tumoralactivityassay

Solublel-Cysdidnotshowanyeffectonbreastcancercellline

MCF-7.Valueswerenotsigniﬁcantlydifferentfromcontrol,with4%

and9%ofgrowthaboveatdosesof1and0.5mg/mLl-Cys,

respec-tively(Fig.2).Onlyanalterationabove30%incomparison with

controlsisconsideredpro-tumorigenic[23].

3.4. Silkcharacterization

3.4.1. Contactangle

Thewettabilityofsilkﬁbroin(SF)wasassessedbythe

measure-mentofthecontactanglebetweenthesurfaceandwaterusing

thesessiledroptechnique.Watercontactanglesmeasuredat10s

showedahydrophilicSFsurface;21.1±1.5◦.ThereforegraftedSF

samplesprovedtobeabletoabsorbexudatesandprovidewound

moisture,whichrenderthemusableashealingwounddressings

againstAD.

3.4.2. Fouriertransforminfraredspectroscopy(FT-IR)

TheinfraredspectraofunmodiﬁedSFisshowninFig.3a.Peaks

at3300cm−1and3100cm−1areassociatedwithamidesAandB,

respectively.At1650cm−1 vibrationsaredue toC Ostretching

vibrationsofamideI,andthepeakat1520cm−1isrelatedtoN H

inplanebendingtogetherwiththeout-of-phaseC Nstretchingof

amideII.

SFcovalentstructuralmodiﬁcationafter25washingcycleswas

alsoassessedbyFT-IR.IRspectraconﬁrmedl-CysbondedonSF

surface,asshownbytheincreaseofpeaksassociatedwithl-Cys

immobilizationonasurface,around3300,3100and1650cm−1,

asshowninFig.3B.AmideIisrelated tovibrationsofthe

pep-tidebonds.However,thepeakat1520cm−1 related toamideII

diminishes,whichtells theinformation ofSFproteinsecondary

structuremodiﬁed/unfoldingbasedontheextentofl-Cys

cross-linkingtoSF.Furthermore3300cm−1 isalsoassociatedwithOH

stretchingrelatedtofree OHinSFaromatictyrosine[24].When

covalent bond formedat Tyr residue thevibration assigned to

OHdecreased.Thismightbethereasonwhythesample

SF-g-l-Cys/Tyr-g-l-CysatpH9.590◦C(spectrume)showedsigniﬁcant

decreaseoftheabsorptionbandat3300cm−1.

3.4.3. Efﬁciencyoffunctionalization/grafting

Theexhaustion efﬁciencyofl-Cys onSFfabricwasassessed

after 3h of functionalization at 90◦C. Allsamples gave rise to

ﬁguresofover80%exhaustionrate,once_l-Cysreadilyadsorbed

toSFchemistry.SF-g-l-Cys/Tyr-g-l-Cysdoublecovalentbonding

showed80.35%(pH5.5)and80.71%(pH9.5),whereasSF-g-l-Cys

singlecovalentbondingrateswere87.09%(pH5.5)and85.94%(pH

9.5).

3.4.4. Tensilestrengthassays

Tensiletestsweredonetoevaluatetheeffectofsinglecovalent

anddoublecovalenttreatmentsonthemechanicalpropertiesof

samples,andareshowninTable2.

AworthymaximumstressandelongationwereobservedforSF

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Fig.3. IRspectraofunmodified(a)andmodifiedsilkfibroinwithdifferentparametersofsurface-activationafter(A)0washing-cyclesand(B)25washing-cycles,with1% owfl-Cysfunctionalization.(b)SF-g-l-CysatpH5.590◦C;(c)SF-g-l-CysatpH9.590◦C;(d)SF-g-l-Cys/Tyr-g-l-CysatpH5.590◦C;(e)SF-g-l-Cys/Tyr-g-l-CysatpH9.590◦C.

Table2

Mechanicalcharacterization.Resistancetoruptureassaysinthreadandstrip.

Material Stressatbreak

(Newton) Elongation(%) Thread SF 31.08±4.06 15.37±4.15 pH5.5SF-g-l-Cys 28.59±4.28 13.69±3.45 pH5.5SF-g-l-Cys/Tyr-g-l-Cys 27.10±5.83 13.99±4.16 pH9.5SF-g-l-Cys 33.69±0.36 18.14±0.02 pH9.5SF-g-l-Cys/Tyr-g-l-Cys 31.15±12.7 16.49±6.72 Strip SF 620.7±54.9 57.49±4.75 pH5.5SF-g-l-Cys 640.6±55.2 59.15±4.47 pH5.5SF-g-l-Cys/Tyr-g-l-Cys 486.6±80.5 55.25±4.37 pH9.5SF-g-l-Cys 566.0±46.3 58.02±8.39 pH9.5SF-g-l-Cys/Tyr-g-l-Cys 709.3±52.2 64.13±6.24

werenot signiﬁcantlydifferentfromcontrols, whichprovesthe covalentmodiﬁcationssetupinthisworkdidnotcompromiseSF.

3.5. Antimicrobialassays

3.5.1. Quantitativemethod(suspension)

SFactivatedsurfacesshowedgoodmicrobialinhibitionresults, asshowninFig.4.Followingthreerinsesafterfunctionalization,no

signiﬁcantdifferences(p<0.05)betweencovalent/doublecovalent

treatmentscouldbeseenregardingmicrobialkilling,exceptthat

allofthemwerehighlymicrobicidal.After24hoursofincubation,

resultsofmicrobialreductionwerecloseto100%.

After5washingcycles,SF-g-l-CystreatmentatpH9.5showed

tobesigniﬁcantly(p<0.05)moremicrobicidalforS.aureus–98.65%

–against 89.33%of SF-g-l-Cys pH5.5.Also, SF-g-

l-Cys/Tyr-g-l-Cysmodiﬁcationswere77.38%forfunctionalizationatpH9.5,and

78.40%forpH5.5.

Ontheotherhand,after25washingcyclesSF-g-

l-Cys/Tyr-g-l-Cysdoublecovalentmodiﬁcationprovedtobeworthwhile,onceit

showedsuchagreatamountofmicrobialreduction,whichproved

toformbonds/structuralconformationsstrongenoughtoresistto

severalwashings.S.aureuswhenadsorbedtoSF-g-

l-Cys/Tyr-g-l-Cys,modiﬁedunderpH9.5with1%owfl-Cys,showedasigniﬁcant

CFUreductionof97.55%(p<0.05),whereaswhenadsorbedto

SF-g-l-Cys bacterialreduction was not soprominent, withresults

averaging67.39%.

3.6. Scanningelectronmicroscopy(SEM)

Foranyconditionafter25washingcycles,therewasadecrease

inthenumberofS.aureusadsorbed.However,forSF-g-

l-Cys/Tyr-g-l-CysfunctionalizedatpH9.5,anoutstandingkillingeffectshowed

almostnobacteriaadsorbedtofabricsurface(Fig.5).

3.7. Transmissionelectronmicroscopy(TEM)

Following25 washing cycles, Fig. 6shows that there was a

ﬁercedegradationofS.aureusmembranes,whenadheredtoSF-g-

l-Cys/Tyr-g-l-Cys,especiallyatpH9.5(Fig.6e).Consideringtheeffect

ofSF-g-l-Cys/Tyr-g-l-CysmodiﬁedatpH5.5(Fig.6c),itshowed

adhered bacteriawith theirmembranes slightly harmed, when

comparingtocontrols.

4. Discussion

Thisworkcompared singlecovalentagainstdouble covalent

immobilizationofabioactiveagentl-Cysteine(l-Cys)onsilkﬁbroin

(SF)knittedfabrictooriginateaneffectiveantimicrobialwound

dressing(bioactivematerial)againstatopicdermatitis (AD)

dis-ease.Furthermore,endapplicationsoflaundryresistanceversus

disposibility,arehereindiscussed.

l-Cys amino acid, which hasa wide spectrum of microbial

activity and well-known safety [10–12], wasfor the ﬁrst time

functionalizedatitslowest concentration– 1%overtheweight

ofthefabric(wof).Inpreviousworkstheminimumamountused

withefﬁciencyof microbialkilling was5%owf,in combination

witheitherwool[23],orcotton[10,12].Thisl-Cysfallis

under-standable,inthatrecentlynewapproachesrelyingonremovalof

lipidsfromthesurfaceofﬁbershaveexposedtheirsurfacewith

avarietyofreactivemoieties.Inthissense,covalenttreatments

canbedirectedstraightforwardtothesereactivesites,ratherthan

physically-directed,whichoffershigherdurabilityandresistance

tolaundry[25].

Forl-CysimmobilizationontoSFfabrictwoprocesseswere

con-sideredinordertopotentiateitsactivity.Theﬁrstprocessrelied

onmodiﬁcationof87%ofSFstructure,namelythecovalent

bond-ingonglycine,alanine,andserineaminoacids.l-Cyswasblended

withSFatbothlowandhighpH’s.ThestrongacidHClplusheat

(90◦C),aswellasstrongbaseNaOHplusheat(90◦C),madeSF

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Fig.5. SEMimagesofadsorbedS.aureusonsilkﬁbroinsurfaceatdifferentconditions.(a)Control;(b)SF-g-l-CysatpH5.590◦_C;_(c)_{SF-g-l-Cys/Tyr-g-l-Cys}_at_pH_5.5₉₀◦_C;

(d)SF-g-l-CysatpH9.590◦C;(e)SF-g-l-Cys/Tyr-g-l-CysatpH9.590◦C.

Fig.6.TEMimagesofdetachedS.aureusonsilkﬁbroinsurfaceatdifferentconditions.Magniﬁcationof10000×.(a)Control;(b)SF-g-l-CysatpH5.590◦_C;_(c)

SF-g-l-Cys/Tyr-g-l-CysatpH5.590◦_C;_(d)_SF-g-l-Cys_at_pH_9.5₉₀◦_C;_(e)_{SF-g-l-Cys/Tyr-g-l-Cys}_at_pH_9.5₉₀◦_C.

esteriﬁcationand“saponiﬁcation”,respectively.Also,thepresence

offurthercarboxylatesinSFisalsodescribed inliterature[26].

Aminogroups ofl-Cys werethen covalentlylinkedbyreacting

withthesecarboxylicacidsbyaminolysis(SF-g-l-Cys).The

sec-ondprocessreliedoncovalentmodiﬁcationoffurther4.8%ofSF

structuraltyrosinearomaticsidechain.Aldehydesoftyrosineside

chainswereblendedwiththeaminesofl-Cys,formingunstable

Schiffbaseintermediates(orimines).Theseintermediateswere

chemicallystabilizedbyreductiveaminationwithNaCNBH3[27],

formingultimatelyacovalentbond.Intheﬁrstprocess,Schiffbase

intermediatesalsoformedatthesesitesbutreverted,oncethey

werenotstabilizedbyreduction.Furthermore,inthisprocessof

graftingl-CysonTyr,importantissuesasTyrlocalization,andits

inﬂuenceonbackboneconformationandpackingwereregarded

[4].l-Cyscarriesathiolategroupinitsmolecularstructurewhich

killsmicroorganismsthroughdisulﬁdebridgeestablishmentwith

somebacterialwallstructuralcomponents[28]oreven

intracellu-larcomponentsifinternalized.

Theabilityof l-Cystoinhibitthegrowthof microorganisms

insolutionwasgivenbyMinimalInhibitoryConcentration(MIC)

studiesofsolublel-CysagainstS.aureuswhichwas5mg/mL.This

valueishighwhencomparedwithchitosanpolymer(0.05mg/mL)

and, onthe otherside, lower than triclosan(6mg/mL) [29,30].

WhenwecomparetheseformerMICresultswithregardofsettling

thesamemolecule(l-Cys)onasurface,datademonstrated

immo-bilizationadvantageouswiththerequirementofloweramountof

l-Cys,namely1%owf(1%overtheweightoftheﬁber(owf)ina

ratioof1gﬁber/50mLsolution;thus,0.2mg/mL).l-Cysmolecules

insolutionbindandgathereachotherthroughS-Sbonds,losing

theirmainreactivethiolates.Ontheotherside,immobilizingl-Cys

ontoasurfacepreventeditsagglomerationandallowedtheuse

oflesserquantitytocoatthesurface,inordertograntSFﬁbers

antibacterialactivity.

It hasalready beendemonstrated thattopical applicationof

antioxidantsdecreasedwoundhealingtime[31].Thetotal

antiox-idantcapacityofl-Cyswasassessedbyusingspectrophotometric

methodswithDPPH◦ (2,2-diphenyl-1-picrylydrazyl)[22].The

l-CysconcentrationthatcausedadecreaseintheinitialDPPH◦by50%

(EC50)wasfoundtobe0,029957␮g/mL.Theseresultsshowedthat

l-Cyshasantioxidantactivity.Sánchez-Moreno[32]showedEC50

valuesofafewotherantioxidants,asgallicacid(26␮g/mL),

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queratin(84_␮g/mL),BHA(93_␮g/mL)andrutin(102_␮g/mL).These

valuesshowthatalbeitl-Cysisantioxidant,itsantioxidantpower

islowbutcanbeincreasedbyrisingthel-Cys concentration,if

necessary(customized).

Theconcentrationofl-CysthatwasusedforSF

functionaliza-tiondidnotinterferewithtumoralcellsgrowthrate,whichproves

thesafetyofusingthisapplicationinneoplasticpatients.

Further-more,l-Cyshasalreadybeenstudiedforitscytotoxicityagainst

ﬁbroblasts,provingtobesafe,hencenotcytotoxic[20].

SF hydrophilic surface, with a contact angle of 21.1±1.5◦,

besidesabsorbingADwoundexudateandprovidingwound

hydra-tionwhichopensthewaytoimportantfactorsforproperhealing,

showedtoattractS.aureuspresentinanADdiseaseenvironment,

allowingS.aureustocontactandbeeliminatedbythegrafted

l-Cys.J.JiandW.ZhangstatedthatahydrophilicsurfaceattractedS.

aureus[33]whereasH.YangandY.Dengindicatedthata

hydropho-bicsurfacereduceditsadhesion[34].

TheformationofsilkstructureisdrivenbyTyrresidues,which

have a strongeffectnot only locally but alsolong-range

inter-molecularinteractions[4].Uponitsdoublefunctionalizationwith

l-Cys,SF structure mighthave adoptedsuchan altered folding

duetocovalentbondingatTyrresidue.Thismightbethereason

whythesampleSF-g-l-Cys/Tyr-g-l-CysfunctionalizedatpH9.5

90◦CshowedsigniﬁcantdecreaseoftheFT-IRabsorptionbandat

3300cm−1.Nevertheless,itispossibletoconcludethatthe

diffus-ingcapacityofl-Cysintheﬁberwashigh,>80%,whichmeansl-Cys

hastheabilitytobindcovalentlytotheﬁbersurfaceeasilyand

strongly.Also,highexhaustionratesmeantalowquantityofl-Cys

requiredtoachieveaneffectiveantimicrobialactivity.

Accordingly,FT-IRresultsconﬁrmedthat_l-Cyswaswell

immo-bilizedtoSFﬁbersatbothpH’soffunctionalization.Furthermore,

as washing cycles were performed, effects at 5 and 25

wash-ingcyclesshowedSF-g-l-Cys/Tyr-g-l-Cysgraftingtobemultiple

washing resistant along with SF-g-l-Cys grafting at pH 9.5. In

accordance withthe chemical surface analyses of SFﬁber

sur-faces,microbiologicdata after24hof incubation,showedfor 5

washingcyclesthehighestkillingofbacteria(98.65%)happened

atpH9.5throughl-Cysgraftedona singlecovalentway

(SF-g-l-Cys)(p<0.05). Thishighmicrobicidalreduction mightbe due

tothestructural/conformationalaccessibilityofl-CysonSFwhen

comparingtoSF-g-l-Cys/Tyr-g-l-Cys.Furthermore,after25

wash-ingcyclestheoptimalbacterialkillingeffect(97.55%)happened

atpH9.5throughdoublecovalentgraftingSF-g-_{l-Cys/Tyr-g-l-Cys}

(p<0.05).Thishighmicrobicidalreductionmightberelatedtothe

exposing of erstwhile structuralfolding-protected l-Cys due to

multiplewashingcycles.Aqualitativeanalysiswasalsoperformed

throughScanning ElectronMicroscopy (SEM) and Transmission

ElectronMicroscopy (TEM) by characterizing S. aureus

accord-ingtoitsshapewhen adsorbedonSFsurfaceafterit hadbeen

washed 25 times (25 washing cycles). Data demonstrated that

few bacteria adhered to the background ﬁbers and the ones

adheredwereinabadshape/morphologyattheconditionof

SF-g-l-Cys/Tyr-g-l-CysfunctionalizedatpH9.5.Asexpected,acidic

conditions of functionalizationyielded poorer grafting of l-Cys

onSFﬁbers.Ontheotherhand,accordingtoHornseyand

Pep-per [35] the higher pH condition of functionalization led to

highereffectiveness ofgrafting [35]once protonatedamines of

l-CysweremoreattractedtotheSFnegativecarboxylategroups

whenunder thealkalinepH9.5. Also, SFhighexhaustion of

l-Cysisduetoitsstructuralreactivity,whichtranslatedinhigher

retentionafterseveralwashingcycles,leadingtoincreased

antimi-crobial properties of SF ﬁbers, when compared to cotton [20].

Mechanical propertiesof SFshowed high,and were not

signif-icantlyalteredwithanytreatments inthis study,whichmeans

SF structure never got compromised by used functionalization

processes.

5. Conclusions

Thisresearchworkdescribesbothsinglecovalentanddouble

covalentgraftingmethodsforthefunctionalizationofsilkﬁbroin

(SF)withantibacterialandantioxidantagentl-Cysteine(l-Cys).The

developedSF-l-Cysmaterialshavethepotentialtoinhibit

medi-callyrelevantS.aureusbacterialspeciesandassuchtobeusedto

controlatopicdermatitissincenotoxicitywasfound.Moreover,the

durabilityofthefabricwassigniﬁcantlyimprovedwhentheactive

antibacterialagentl-CyswasdoublecovalentlyattachedtoSF

tyro-sine.Itwasdemonstratedtheeffectivenessofadoublecovalent

grafting,withtheimportanceofSFtyrosine(Tyr)covalent

link-ingwithl-Cys(SF-g-l-Cys/Tyr-g-l-Cys)forseveralwashingcycles,

whereasforadisposableapplicationasinglecovalentmechanism

ofgraftingl-Cysprovedtobesufﬁcient(SF-g-l-Cys).

Acknowledgments

TheauthorswouldliketoacknowledgetheFundac¸ãopara a

CiênciaeTecnologia(FCT)forthefundinggrantedconcerningthe

project – PTDC/EBB-BIO/113671/2009

(FCOMP-01-0124-FEDER-014752) Skin2Tex and Fundo Europeu de Desenvolvimento

Regional(FEDER)throughCOMPETE–ProgramaOperacional

Fac-toresdeCompetitividade(POFC)fortheco-funding.

TheauthorswouldalsoliketoacknowledgetheFundac¸ãopara

a Ciência eTecnologia (FCT)for the fundingfor the PhD grant

SFRH/BD/91444/2012.

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