Synthesis of Antifungal Agents from Xanthene and Thiazine Dyes and Analysis of Their Effects

14 Pages·2016·3.99 MB·English

nanomaterials
Article
Synthesis of Antifungal Agents from Xanthene and
Thiazine Dyes and Analysis of Their Effects
JooRanKim1andStephenMichielsen2,*
1 FiberScience&ApparelDesign,CornellUniversity,Ithaca,NY14850,USA;[email protected]
2 TextileEngineering,ChemistryandScience,NorthCarolinaStateUniversity,2401ResearchDr.,Raleigh,
NC27695,USA
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:ThomasNann
Received:10November2016;Accepted:9December2016;Published:20December2016
Abstract: Indoorfungigrowthisanincreasinghomehealthproblemasourhomesaremoretightly
sealed. Onethingthatlimitsdurabilityoftheantifungalagentsisthescarcityofreactivesiteson
many surfaces to attach these agents. In order to increase graft yield of photosensitizers to the
fabrics,poly(acrylicacid-co-styrenesulfonicacid-co-vinylbenzylrosebengalorphloxineB)were
polymerizedandthengraftedtoelectrospunfabrics. Inanalternativeprocess,azureAortoluidine
blueOweregraftedtopoly(acrylicacid),whichwassubsequentlygraftedtonanofiber-basedand
microfiber-based fabrics. The fabrics grafted with photosensitizers induced antifungal effects on
all seven types of fungi in the order of rose bengal > phloxine B > toluidine blue O > azure A,
whichfollowsthequantumyieldproductionofsingletoxygenforthesephotoactivedyes. Their
inhibitionratesforinactivatingfungalsporesdecreasedintheorderofP.cinnamomi,T.viride,A.niger,
A.fumigatus,C.globosum,P.funiculosum,andM.grisea,whichisassociatedwithlipidcompositionin
membraneandthemorphologyoffungalspores. Theantifungalactivitywasalsocorrelatedwiththe
surfaceareaoffabrictypeswhichgraftedthephotosensitizercovalentlyonthesurfaceasdetermined
bytheboundcolorstrength.
Keywords: antifungalphotosensitizer;nanofiber;Aspergillus;Chaetomium;Magnaporthe
1. Introduction
Antimicrobialfabricsbasedonnaturalorsyntheticpolymerstreatedwithsilverionsandcopper
nanoparticlehavebeenusedoften[1,2]. Recently,4-amide-piperidine-C12hydrogelcoatedsilicone
films exhibited antimicrobial effects on both Gram-negative, Gram-positive bacteria and fungi [3].
Poly(methylmethacrylate)coatedpoly(2-hydroxyethylmethacrylate)filmshadbeenreportedtohave
anantimicrobialeffectonCandidaalbicansforupto28days[4]. However,thesegraftedchemicals
havebeenreportedtoproduceadverseeffectsonhumans,resultinginanaccumulationinthehuman
skin. Silvercandiffuseintotheskin,darkenuponexposuretosunlight,resultinginablueorgray
discolorationoftheskin[5]. Hence,non-hazardousdyematerialssuchasphotosensitizers(PS)have
been investigated intensively for the replacement of silver and copper, and hazardous chemicals.
Inparticular, xanthenedyessuchasrosebengal, phloxineB,anderythrosinehavebeenshownto
exhibit antimicrobial activity and are considered safe [6], since they are commonly used as dyes
in the food, cosmetics and textiles where they are approved as safe color additives in the USA,
theEuropeanUnion(EU)andJapan[7]. Furthermore,theyarelessexpensive,havebettercoloring,
andareeasiertoproducethannaturaldyes[8]. MethyleneblueandtoluidineblueOhavealsobeen
commonlyusedassafePS[9]. AzureAandAzureAeosinatehavebeenreportedtoinhibit60%of
thegrowthinhibitionofC.albicans. Furthermore,variousPShavebeenshowntobeeffectiveagainst
fungiandyeastsuchasAspergillusfumigatusandKluyveromycesfragilis,Kluyveromycesmarxianusand
Nanomaterials2016,6,243;doi:10.3390/nano6120243 www.mdpi.com/journal/nanomaterials
Nanomaterials2016,6,243 2of14
C.albicans[10]. HongandGanghavestudiedantimicrobialpropertiesusingrosebengalincorporated
intopolyurethane-coatedleatherbyapaintingmethod[11]. However,therearefewerstudiesusing
PSgraftedtopolymericmaterials. Theselimitationshaveaffectedtheutilizationofphotodynamic
actionsinvariousapplications.
Photodynamictherapy(PDT)isatreatmentwithPS,whichcanbeactivatedbyexposuretovisible
lightinaspecificwavelengthrange. PShasastableelectronicconfiguration,whichisinasingletstate
intheirlowestorgroundenergylevel,S whichmeansthattherearenounpairedelectronspins[12].
0
Uponabsorptionofaphotonwithinaspecificwavelengthrange,amoleculeispromotedtoanexcited
state,S orS ,withhigherenergy[13]. TheexitedsingletstateofPScanreturntothegroundstateby
1 2
emittingaphotonaslightenergy,i.e.,byfluorescence. Alternatively,themoleculemayconverttothe
tripletstate,T orT ,viaintersystemcrossing,whichinvolvesachangeinthespinofanelectron[14].
1 2
Thetripletgroundstateofoxygenexchangesspinwiththetripletexcitedstateofthedyetoreturnthe
dyetoitssingletgroundstateandtoproducesingletoxygenatthesametime.
Singletoxygen(1O )isthefirstexcitedelectronicenergystateofmolecularoxygenspecies. Itis
2
oneofthemostactiveintermediatesinvolvedinchemicalandbiochemicalreactions. Hence,1O can
2
reactwithmanykindsofbiologicalmolecules,suchasDNA,proteinsandlipids,resultinginchemical
reactionsduetotheproductionofthisreactiveoxygenspecies[15].
Aspergillus niger, Aspergillus fumigatus, Trichoderma viride, Penicillium funiculosum, and
Chaetomium globosum are the most common indoor fungal species that are opportunistic human
pathogensresultinginaspergillosisandpneumocytosisinimmunosuppressedpatientsandallergy,
rhinitisinhealthyhumans[16,17]. MagnaporthegriseaandPhytophthoracinnamomi(Oomycetes)are
plantpathogensinducing,respectively,riceblastresultinginseriouscroplossglobally[18]androot
rotandcankeronEucalyptus[19].
Inthisstudy,photosensitizersrosebengal(RB),phloxineB(PB),azureA(AA),andtoluidine
blueO(TBO),wereeithercopolymerizedwithacrylicacidorgraftedtopoly(acrylicacid),andthen
graftedtofabricsconsistingofnanofiber-basedfabricsormicrofiber-basedfabrics. Theantifungal
effectsofthefabricswerethenevaluatedagainstseventypesoffungiandanalyzedantifungaleffect.
2. MaterialsandMethods
2.1. Materials
Poly(acrylicacid)(PAA,averageM ~450,000),4-vinyl-benzylchloride,4-styrenesulfonicacid,
V
acrylic acid (AC), nylon 6,6 pellets, formic acid (reagent grade > 95%), phloxine B (PB), azure A
(AA),toluidineblueO(TBO),potatodextroseagar(PDA),potatodextrosebroth,phosphatebuffer
saline (pH 7.0), Tween® 20, RPMI 1640 medium, sterile water, 3-(N-morpholino)-propanesulfonic
acid (MOPS), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)
were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). RB and meltspun
nylon6.6,CerexSpectramax®weredonatedbyLaamScience(Cary,NC,USA),while0.5McFarland
standardforturbiditymeasurementswaspurchasedfromFisherScientificCo. (Pittsburgh,PA,USA).
Aspergillusniger(ATCC6275),Trichidermaviride(ATCC28020),Penicilliumfuniculosum(ATCC10509),
Aspergillus fumigatus (ATCC 13073) and Chaetomium globosum (ATCC 6205) were donated from
the United States Department of Agriculture, Agricultural Research Service, Peoria, IL, USA.
MagnaporthegriseaandPhytophthoracinnamomi(fungus-likeorganism)weredonatedfromtheNorth
CarolinaStateUniversity,PlantPathogensDepartment,Raleigh,NC,USA.
2.2. PreparationofNanofiber-BasedFabrics
Nanofiber-basedfabricwaspreparedusingelectrospinning,whichproducedfibersintherange
from200nmto5µmindiameterasshownFigure1a. Ahighvoltage(20kV)wasappliedbetween
needletipandrollercollector. First,18%w/vnylon6,6pelletsweredissolvedintoformicacidand
Nanomaterials2016,6,243 3of14
agitatedat70◦Cfor8h. Thesolutionwasputintoa10-mLsyringeandthenplacedonthepump.
Thefeedingrateofsolutionwas1mLperhour.
Nanomaterials 2016, 6, 243  3 of 13 
Nanomaterials 2016, 6, 243  3 of 13 

Figure 1. Nanofibers using electrospinning using roller collector, (a) nylon 6,6 fabric consisting of 
Figure 1. Nanofibers using electrospinning using roller collector, (a) nylon 6,6 fabric consisting
nanofibers with average diameter 505 nm (σ = 152.5 nm); and (b) melt spun microfibers (Cerex 
of nanofibers with average diameter 505 nm (σ = 152.5 nm); and (b) melt spun microfibers
Spectramax® nylon 6,6) with diameter 17.3 μm (σ = 0.86 μm). 
(CerexSpectramax®nylon6,6)withdiameter17.3µm(σ=0.86µm).

2.3. Preparation of Water‐Soluble Photosensitizers 
Figure 1. Nanofibers using electrospinning using roller collector, (a) nylon 6,6 fabric consisting of 
2.3. PreparationofWater-SolublePhotosensitizers
The  xannatnhoefinbeer s wdiythe sa ve(rRagBe  doiarm etPerB 5)0 5 wnmer e(σ  =i n1c52o.5r pnomr)a; taendd  (bi)n mtoe lt pspoulny (maiccrroyflibice rs a(Cciedre)x   (PAA)  via 
Spectramax® nylon 6,6) with diameter 17.3 μm (σ = 0.86 μm). 
Tpholeyxmaenrtizhaetnioend. yBerisef(lRyB, 3o gr oPfB R)Bw oerr e2.i5n gc oorfp PoBr awteads sitnirtroedp oinly a( a7c0r‐ymliLc saocliudt)io(nP AofA 5)0v:5i0a vp/vo ldyimstiellreizda tion.
Brieflwy,a3tegr/aocf2e.t3Ro. BPnreeo paranr2adt.i 5o0n.g 5o1f oW mfaPtLerB ‐oSfwo l4ua‐bvlsei nPsythiolrt‐orbseeendnsziityinzle racsh 7lo0r-imdeL ast o6l5u °tCio fnoro 3f 5h0. :T5h0e vp/rvecdipiisttaitlele, dvinwyal tbeern/zaycl‐etone
and0x.a5n1thmeLneo dfy4Te- hv(eVi nBxyXanlD-thb weennhe zerdyeyl eXcs Dh(l RorBerf iedorres  taPoBt t)6h 5ew x◦erCaen tfihonceronr3ep ohdr.yateTe dhR eBin potorr  ePpcBoi)ply, i(wtaacartyse lfi,civl tieancrieydd)l  ab(nPeAdnA zw)y alvs-ihxaea dn.t Nheenxte dye
(VBXtDhew VhBeXrepDoX lwyDmaesrr eidzfaiestrisosonlt.v oBerdtieh fileny, x 3wa gna ottefh rRe Ban noedr d2 a.y5c ege tRoofnB PeBo  awrta PtshB set)i ,rvrweodla uisnm fiae l7 t0re‐amrteiLod  sooalfnu 1tdi:o1nw  aoanf s5dh0 :pe50do vl.y/vNm deeisxrtiitzllteehdd e wVitBhX 4D‐ was
styrene swualfteorn/aicce atocnide  a(nSdA 0A.5)1 a mnLd  oafc 4r‐yvlinicy la‐cbiedn z(yAl Cch)l oarti d1e:4 a0t :6154 °0C m foorl a3 rh r. aTthieo  porfe cVipBitXaDte,:  vSiSnAyl :bAeCnz tyol‐ increase 
dissolvedinwaterandacetoneatthevolumeratioof1:1andpolymerizedwith4-styrenesulfonicacid
water soxluanbtihlietnye  tdoy eth (Ve BpXoDly wmheerres  XaDn dre tfoer sc oton tfheer  xfaunnthcetnioen dayle g RrBo uorp PsB w), iwthas w filhteircehd  taon dg rwaafst hteod n. Nyleoxnt  [20,21]. 
(SAA)andactrhye lVicBaXcDi dwa(sA dCis)soalvte1d: 4in0 :w14at0erm anodl aarcertoantieo ato tfhVe vBoXluDm:e SrSatAio :oAf C1:1t oanidn pcorelyamseeriwzeadt weriths o4l‐ubilitytothe
After copolymerization, poly(acrylic acid‐co‐styrene sulfonic acid‐co‐vinyl benzyl rose bengal or 
polympehrlosxainneds tytorBenc) eo snuwflfeeornreifcu  ancoicdbt i(tSoaAninAae)l dagn. rdo auAcrp ysliccw oacniitddh (eAwnCshi) niacgt h1 :4t0oa:1gg4e0rn amtf,o tlatro4 r‐an(t4iyo,6l oo‐fdn VimB[2Xe0Dt,h:2 So1Sx]Ay.:‐AA1C,f3 tt,eo5r ‐intcrcioraepzasoinel ‐y2m‐yel)r‐i4z‐ation,
water solubility to the polymers and to confer functional groups with which to graft to nylon [20,21]. 
poly(macertyhlyilcmAaocfrtiepdrh -cocoolpi-noslityuymmree rcnizhealtoisorunid,l fepo o(nDlyi(McacaTrycMliicdM -ac)c,oi dw-‐vcaois‐ns utyysrleebndee t nosu zplyfroolnmrico oastceeid cb‐hceoen‐vmginiacylal lob rerenapzcyhtl iloroonxssei  nbbeeetnwBga)ele wonre  –rCeOoObHta ined.
Acongdroeunpsisn ogpfh PalogAxeiAnne ot ,f 4Bp-)o( 4ly,w6me-rdeeri imzoeebdtt ahdinoyexed ys. o-1lAu,3 t,i5oc-notn rtdoiae NnzsiHinng-2 2 e-nyadgl‐)eg-n4rt,-o mu4pe‐s(t4 ho,6yf‐ dlnmiymloeotrnhpo 6hx,y6o‐ 1lfia,3nb,5iru‐itcrmisa ztocinh p‐2lro‐oyrld)i‐du4‐ece( aDmMidTeM  M),
wasulisnekdagtoesp maresot hsmhyloomwtoernpc hihnoe lFminiigiucumar lech r2leo.a ricdtei o(DnMsTbMeMtw), eweans u–sCedO toO pHromgortoe uchpesmoicfalP rAeaActioonfs pbeotwlyemene –rCiOzeOdH dyesolution
groups of PAA of polymerized dye solution to NH2 end‐groups of nylon 6,6 fabrics to produce amide 
toNH end-groupsofnylon6,6fabricstoproduceamidelinkagesasshowninFigure2.
2 linkages as shown in Figure 2. 

Figure 2. The grafting scheme of poly(acrylic acid‐co‐styrene sulfonic acid‐co‐vinyl benzyl rose bengal 
Figure2.Thegraftingschemeofpoly(acrylicacid-co-styrenesulfonicacid-co-vinylbenzylrosebengal
or phloxine B) or thiazine dyes grafted with poly(acrylic acid) (PAA) to the nylon fiber forming   
orphloxineBr)anodrotmh icaozili snheapdey. Des isg proalfytmederiwzeidt hdype omlyol(eaccurley sluiccha acsi dp)ol(yPmAerAiz)edto xatnhtehennye ldoynesfi obr ethriafzoirnme ingrandom
Figure 2. The grafting scheme of poly(acrylic acid‐co‐styrene sulfonic acid‐co‐vinyl benzyl rose bengal 
coilshape.Dispolymerizeddyemoleculesuchaspolymerizedxanthenedyesorthiazinedyesgrafted
or p hloxine B) or thiazine dyes grafted with poly(acrylic acid) (PAA) to the nylon fiber forming 
withPAA.ForRB,R andR areIandCl.ForPB,R andR areBrandClandTBOhasmethylgroup
random coil shap1e. D is p2olymerized dye molecule1 such as2 polymerized xanthene dyes or thiazine 
atR .RoseBengal=RB;phloxineB=PB;toluidineblueO=TBO.
8
Nanomaterials2016,6,243 4of14
The thiazine dyes were also grafted to PAA as follows. A 5% w/v PAA solution in distilled
waterwaspreparedatroomtemperature;100µmol/LofAAorTBOwasaddedtothePAAsolution.
Afterstirringfor1h, 0.3gDMTMMwasaddedtothemixture[22]. Afterstirringforanother3h,
thethiazinedye-grafted-PAAsolutionwasproducedasshowninFigure2.
2.4. GraftingofPhotosensitizerstoFabrics
Inordertograftthephotosensitizercontainingpolymerstothefabric,eachpolymerizedsolution
was prepared at 100 µmol/L concentration in a flat-bottomed dish, which had been covered with
aluminumfoiltoexcludelight. Electrospunnanofiber-basedfabricsandmicrofiber-basedfabricswere
immersedintothesolutionbath. Thecondensingagent,0.3gDMTMM,wasdissolvedinthesolution
bath. After12h,thefabricwasremovedfromthesolutionandplacedintoanoven(WernerMathis
AGLTF134489,Concord,NC,USA)for1minatthetemperatureof170◦C.
2.5. PreparationofInoculum
A.fumigatus,A.niger,T.viride,andP.funiculosum,C.globosum,P.cinnamomiandM.griseawere
culturedat35◦Conpotatodextroseagar(PDA)platesforsevendays. Brothmedium,RPMI-1640
whichcontains0.2%glucoseand0.165mol/LMOPS(3-N-morpholinopropanesulfonicacid)atpH7.0,
wasusedasmediumforfivefungi,whilethepotatodextrosebroth(PDB)wasusedasmediumfor
P. cinnamomi and M. grisea. Next, 10 mL broth medium with 0.01% wetting agent Tween® 20 was
addedtotheculturesandthenscrapedtoseparatespores. Thescrapedsolutionintheculturewas
filteredthroughMillipore®polytetrafluoroethylenefilmtoremovehyphae.
2.6. InhibitionZoneTest
The inhibition zone test of fabrics grafted with photosensitizers RB, PB, TBO and AA was
performedasfollows. A100µLinoculumofeachstrainpreparedaboveat2 × 106 CFU/mLwas
transferredtoaPDAplateandthena5×5cmpieceofthefabricspreparedabovewereplacedonto
theinoculatedPDAplate. Theplateswereplacedintoatrayandthetraywasplacedunderthelamp.
Awater-filledpyrexglassdishwasplacedbetweenthetrayandthelamptoabsorbinfraredlightfrom
aphotofloodlamp(SmithVictor,Griffith,IN,USA)toavoidheatingthetray. Thelampwasplaced
35cmabovetheglassdishandtheinoculatedPDAplateswereplacedbelowtheglassdish. Thelight
intensitywasmeasuredusingadigitalilluminancemeter(ModelLX1330B,UnionCity,CA,USA)at
15,500Lux. Theplateswerekeptunderthisexposurefor5hat24◦Candthenincubatedforseven
daysinthedarkat35◦C.
2.7. MinimumInhibitoryConcentration—BrothMicrodilutionTest
In order to test the effectiveness of the free photosensitizer dyes, minimum inhibitory
concentration(MIC)wasconductedaccordingtotheCLSIM38-Astandard[23];100µLof500µmol/L
aqueoussolutionsofRB,PB,AA,andTBOsolutionsweretransferredtothefirstwellsinthe96-well
plates. Then each column was diluted by two-fold up to 0.98 µmol/L. The spore concentration
was adjusted to lie in the range of 2 × 106 CFU/mL using 0.5 McFarland standard as a reference
and a hemocytometer (Hausser Bright-Line and Hylite Counting Chambers, Horsham, PA, USA).
Then100µLinoculumoffungalsporeswasdepositedintoeachwellcontainingthefreephotosensitizer
solutionspreparedabove. The96-wellplateswerethenilluminatedfor3hat24◦C.Finally,a96-well
platewasincubatedat35◦Cforallfungifor48h. AlltestswereconductedthreetimesandthenMIC
wasselectedasthehighestconcentration.
2.8. QuantitativeAntifungalAssay
A quantitative antifungal assay was also conducted following a modified ASTM E2149-01
method [24]. The inoculum was diluted into sterile phosphate buffer solution (pH 7.0) to obtain
Nanomaterials2016,6,243 5of14
afinalconcentrationof2×105CFU/mL.Thepreparedinoculum(50mL)wasaddedintoeach125mL
Erlenmeyerflaskandthentreatedoruntreatedcircularfabricsampleswith4.8cmindiameterwere
immersedintheflask,whichwasthencoveredwithacapwithaholeinit. Theflaskswereplaced
underthelightasabovefor3hat24◦Candtheflaskwasshakenat200strokes/minutethroughout
the test. Then a100 µL aliquot of inoculum from each flask was transferred to individual wells of
a96-welltrayevery30min;96-welltrayswereincubatedinthedarkat35◦Cfor48h. Theoptical
density(OD)wasmeasuredat405nmusingBiotek® SynergyHTmulti-modereader. TheODsof
thetreatedfabricwithinoculum(B)wassubtractedfromtheODsoftheuntreatedfabric(A)with
inoculum[24,25]. Thefinalreductionpercentagewascalculatedasfollows:
A−B
×100 (1)
A
where A is the OD at 405 nm of the solution in the well containing the inoculum exposed to the
untreatedfabricsampleandBistheODofthesolutioninthewellcorrespondingtoeachtreatedfabric
afterthespecifiedcontacttimebetweenthetreatedandilluminatedfabricsample.
2.9. Characteristics
Thespecificsurfaceareasofuntreatednanofiber-basedfabricandmicrofiber-basedfabricswere
obtained from the Brunauer-Emmett-Teller (BET) analysis using liquid nitrogen as the adsorbate
and a Gemini VII 2390p physiosorption analyzer from Micrometrics coupled with SmartPrep 065
degassing unit (Micromeritics Instrument Co., Norcross, GA, USA). The chopped fabric sample
(5×5cm) consisting of nanofibers (0.109 g) or microfibers (0.249 g) was used for BET analysis.
Fieldemissionscanningelectronmicroscopy(JEOL6400)(JEOLUSAInc.,Peabody,MA,USA)was
used to examine the morphology of spores from the seven pathogens and to examine the nylon
fibers. The amounts of RB, PB, AA and TBO grafted to the nanofiber-based and microfiber-based
fabrics were obtained from reflectance measurements over the spectral range 360–700 nm using
aDatacolorSF600X(Lawrenceville,NJ,USA)spectrometer. Thefabricswerefoldedtoensureoptical
opacity, whichrequiredatleast16layersthickformicrofiber-basedfabricsandatleastfivelayers
for nanofiber-based fabrics. Each measurement was repeated two times in four different spots of
thesamples.
3. ResultsandDiscussion
Antifungal agents must be on the surface of fibers in order to be able to inhibit the microbes.
However,typicaltreatmentswithantifungalagentsoftenwashoffeasily. Thus,itisadvantageousto
permanentlyattachtheantifungalagents. Theaminogroupsofnylon6,6canreactwithcarboxylicacid
groupsfromthepolymerized,PAA-basedphotosensitizerdyes. Thisenabledthepermanentgrafting
ofthephotosensitizerdyesthathadbeeneithercopolymerizedwithAC-(xanthenedyes)orthathad
beengraftedontoPAA-(thiazinedyes),thusproducingamidelinkages.
3.1. SpecificSurfaceAreaofFabricsandColorStrength
Figure1a,bshowstheSEMimagesoftheelectrospunnanofibersandthemelt-blownmicrofibers.
The diameters of the fibers were measured from the SEM images. The diameters of two types of
fabricswerecalculatedbyrandomlyselecting30microfibersandmorethan100nanofibersunderthe
SEMimages. Theaveragediameterofthenanofiberswas505nm(σ=152nm), whilemicrofibers
hadanaveragediameterof17.3µm(σ=0.86µm). Thespecificsurfaceareawasmeasureddirectly
usingBETanalysis,givingaspecificsurfaceareaof28.8m2/gforthenanofiber-basedfabric,whilethe
microfiber-basedfabrichadaspecificsurfaceareaof1.41m2/g. Thus,theBETanalysisgivesaspecific
surfacearearatioof20:1forthenanofiber-basedfabricstothemicrofiber-basedfabrics.
Nanomaterials2016,6,243 6of14
The amount of photosensitizer dye on the fabric surface should be proportional to the color
strengthofthefabric. ThefabriccolorstrengthwascalculatedusingreflectancepercentageandK/S
valueasshowninEquation(2)[26].
K (1−R∞)2
= = F(R∞) (2)
S 2R∞
whereKandSareabsorbanceandscatteringcoefficients(cm−1),andR∞ isthereflectanceforinfinite
thickness. If the fabric is sufficiently thick so that it is opaque, Rλ = R∞, where the reflectance is
measuredatthewavelengthofmaximumabsorbance[27].
TheK/Svalueswereconvertedtorelativecolorstrength(%)usingEquation(3)[28].
K/Softreatedfabric
RelativeColorStrength(%) = ×100 (3)
K/Sofuntreatedfabric
Table1givesboththeK/Svalueandthecolorstrengthsforeachofthefabricsusedinthisstudy.
TheresultsinTable1implythatthereis6.6to12timesmorerelativecolorstrengthormoreamountof
dyeonthenanofiber-basedfabricsthanonthemicrofiber-basedfabrics.
Table1.Reflectancepercentages,K/Svaluesandcolorstrength(%)ofnanoandmicrofabricsgrafted
withRB,PB,TBO,andAA.
Reflectance%atλmax F(R∞) ColorStrength(%)
GraftingDye
Nano Micro Nano Micro Nano Micro
RB 2.93±0.13 21.2±0.86 0.16 0.015 60.1 5.0
PB 3.07±0.21 20.7±1.23 0.15 0.015 51.2 5.0
TBO 4.18±0.19 21.5±1.01 0.11 0.015 36.7 5.0
AA 4.94±0.16 22.8±0.97 0.09 0.014 30.7 4.7
3.2. AntifungalActivity—MinimumInhibitoryConcentration(MIC)Test
Before testing the antifungal activities of fabrics with the photosensitizer dyes attached,
theantifungaleffectivenessofthefreedyeswastestedusingtheMICtest. TheresultsshowninTable2
indicatethatforRBtherewasnovisibleturbidityorgrowthat62.5µmol/Lwhileconcentrationsbelow
62.5µmol/LshowedvisiblehyphalgrowthonA.nigerandA.fumigatus,P.funiculosum,andC.globosum.
For T. viride, RB displayed the MIC of 31.2 µmol/L and 15.6 µmol/L for P. cinnamomi. For PB,
P.funiculosum,andC.globosumshowedthelargestresistancewithMICat125µmol/L,whileA.niger
andA.fumigatushadMICat62.5µmol/L.TBOandAAbothhadmuchhigherMICsthanRBorPB.
TBOhasanMICof62.5µmol/LonA.niger,A.fumigatus,andT.viride. ForAA,below125µmol/L
visiblegrowthandturbiditywasobservedforA.nigerandA.fumigatus,P.funiculosumandC.globosum,
butnotT.viride,givingMICof62.5µmol/L.RB,PB,TBO.AAshowedthelowestMIConT.viride
whileexhibitingthelargestMIConP.funiculosumorC.globosum.
Table2.Theminimuminhibitionconcentration(MIC)ofRB,PB,AA,andTBOtopreventthefungal
growthinbrothmicrodilutiontestsbyvisualobservation(thehighestconcentrationtoinhibitgrowth
wasselectedamongthreetests).
MIC(µM)
GraftingDye
M.grisea P.funiculosum C.globosum A.niger A.fumigatus T.viride P.cinnmomi
RB 125 62.5 62.5 62.5 62.5 31.2 15.6
PB 250 125 62.5 62.5 125 31.2 31.2
AA 250 125 62.5 125 62.5 62.5 62.5
TBO 250 125 125 62.5 62.5 62.5 62.5
Nanomaterials2016,6,243 7of14
For P. cinnamomi, the MIC was very low, with an MIC of 15.6 (RB) and 31.25 (PB) µmol/L.
However, M. grisea was quite resistant to inhibition with an MIC of 125 µmol/L for RB while PB
Nanomaterials 2016, 6, 243  7 of 13 
inhibitedM.griseaat250µmol/L.AAandTBOinhibitedfungalgrowthofM.griseaabove250µmol/L
andP.cinnFaomr oPm. iciantn6am2.o5mµi,m thoel/ ML.ICM w.garsi sveearsyh loowwe, dwtithhe alanr gMeIsCt roefs i1s5t.a6n (cReBt)o apnhdo 3t1o.s2e5n (sPitBi)z eμrmsoshl/oLw.  ing
thehiHghoewsetvMerI, CMa. tg2ri5s0eaµ wmaos l/quLi.te resistant to inhibition with an MIC of 125 μmol/L for RB while PB 
inhibited M. grisea at 250 μmol/L. AA and TBO inhibited fungal growth of M. grisea above 250 μmol/L 
3.3. Aanntdif uPn. gcianlnAamctoimviit ya—t 62In.5h μibmitoiol/nLZ. Mon. egrTiesesat showed the largest resistance to photosensitizers showing 
the highest MIC at 250 μmol/L. 
To evaluate the antifungal effectiveness of the surface-bond photosensitizers, two types of
antifungalassayswereconductedagainstsevenfungitypes. Thefirstmethodexaminedvisiblegrowth
3.3. Antifungal Activity—Inhibition Zone Test 
ofhyphaeandgerminationonthefabrictoidentifythezoneofinhibition. Thistestisaqualitativetest
To evaluate the antifungal effectiveness of the surface‐bond photosensitizers, two types of 
thatreliesontheabilityoftheantifungalagenttodiffuseawayfromthetreatedfabricincombination
antifungal assays were conducted against seven fungi types. The first method examined visible 
withtheantifungalactivityoftheagent. Largeinhibitionzonesindicatethattheagentisquitemobile
growth of hyphae and germination on the fabric to identify the zone of inhibition. This test is a 
whilesmallinhibitionzonesindicateeitherlittlemobilityoralackofeffectiveness.
qualitative test that relies on the ability of the antifungal agent to diffuse away from the treated fabric 
Finig cuormeb3inasthioonw wsithin thheib aintitoifnunzgoaln aecstivfiotyr o(fa t)heP a.gceinntn. Lamarogme iin;h(ibbi)tioTn. zvoinriedse i;ndaincadte (tch)at Mth.e aggreisneta  on
nanofiisb qeur-itbea mseodbilaen wdhimle iscmroafillb inerh-ibbaitsioend zfoanbersi icnsd.icaInte egiethneerr laitlt,let hmeobfialibtyri ocsr ae lxahckib oift eedffelcetisvsengersosw.  th of
P.cinnamoFmigiuarned 3 Tsh.ovwirsid inehoinbittihoen szuornfeasc feo,rc (oa)m Pp. acirnendamtoomthi; e(bg)r To.w vitrhidoe;f aMnd. (gcr)i Msea. .grTisheae onna nnaonfiobfiebre-rb‐ased
fabricbsagserda fatnedd mwicitrhofiRbBer‐abnasdedP fBabsrhicosw. Ine dgennoeraglr, othwet fhaborficPs .exchinibniatemdo lmesis ogrroTw.tvhi roifd Pe. ocinnntahmeomfaib arnicd, but
nanofiTb. veirr-ibdea soend thfea bsurircfascge,r caoftmedpawreidth toA thAe gshroowwthe dofP M.c. ignrnisaema. oTmhei gnaronwofitbhera‐rboausendd fathbreicesd ggreafotefdt hweitfha bric
RB and PB showed no growth of P. cinnamomi or T. viride on the fabric, but nanofiber‐based fabrics 
andsubstantialgrowthofT.virideonthefabric. Themicrofiber-basedfabricwithRBandPBshowed
grafted with AA showed P. cinnamomi growth around the edge of the fabric and substantial growth 
littleP.cinnamomiandT.viridecolonygrowthonthefabricwhilethecontrolwascompletelycovered
of T. viride on the fabric. The microfiber‐based fabric with RB and PB showed little P. cinnamomi and 
bygrowthandgermination. Mostmicrofiber-basedfabricsgraftedwithAAandTBOexhibitedno
T. viride colony growth on the fabric while the control was completely covered by growth and 
inhibitionoffungalgrowth(notshown). Ontheotherhand,M.griseaexhibitedsubstantialgrowth
germination. Most microfiber‐based fabrics grafted with AA and TBO exhibited no inhibition of 
onallfufanbgrailc gsreoxwctehp (tnfoot rshthoewRn)B. -Ognr atfhtee dothnearn hoafinbde, rM-b. agsreisdeaf eaxbhriibci.teFdig suurbest3anatlisaol gsrhoowwths otnh aatllt fhaebrziocsn eof
inhibietxiocenpfto frora ltlhter ReaBt‐egdranftaendo nfiabneorfi-bbears‐ebdaseadn dfambriicc.r oFifigbuerre- 3b aaslseod sfhaobwrisc tshwata tshen ezgonlieg iobf lienhailbthitoioung fhort here
wasvaelrl ytreliatttelde nfuannogfiibgerro‐bwasthedo anndth meicfarobfribicesr‐tbraesaetde dfabwriictsh wRaBs noergPligBi.bElev aeltnhothuoghu gthherTeB wOasa vnedryA liAttlew  ere
lessefffuencgtii vgero,wthteh zoonn theeo ffaibnrhicisb tirteioatnedw waisths tRilBl nore gPlBig. Eibvleen. tThhoiusgihs TinBdOi raencdt AevAid weenrcee letshsa etfftehcetivaen,t tihfue ngal
zone of inhibition was still negligible. This is indirect evidence that the antifungal photosensitizers 
photosensitizerswereindeedgraftedtothefabricsurfaces. Theinhibitionzoneisexpectedtobenearly
were indeed grafted to the fabric surfaces. The inhibition zone is expected to be nearly zero if the 
zeroifthephotoactivepolymeriscovalentlyattachedtothefabricsincetheactiveantifungalagentis
photoactive polymer is covalently attached to the fabric since the active antifungal agent is believed 
believedtobesingletoxygen,whichhasashortlifetime,limitingdiffusiontoaround0.1mm[29]and
to be singlet oxygen, which has a short lifetime, limiting diffusion to around 0.1 mm [29] and thus 
thuslimitingantifungalactivitytocloseproximitytothepolymerizedphotosensitizers.
limiting antifungal activity to close proximity to the polymerized photosensitizers. 

FigureFig3u.rTe h3e. Tihneh iinbhitiibointiozno znoenete tsetsto off nnaannoo aanndd mmicircor ofabfaribcr gicragftreadf twedithw RiBth, PRBB, ,APAB a,nAdA TBaOn donT (Ba)O  on
P. cinnamomi; (b) T. viride; and (c) M. grisea, The 1st column is the control, the 2nd is the nano fabric 
(a)P.cinnamomi;(b)T.viride;and(c)M.grisea,The1stcolumnisthecontrol,the2ndisthenanofabric
grafted with RB, the 3rd is the nano fabric grafted with PB, the 4th is the micro fabric grafted with RB, 
graftedwithRB,the3rdisthenanofabricgraftedwithPB,the4thisthemicrofabricgraftedwithRB,
the 5th is the micro fabric grafted PB, the 6th is the nano fabric grafted with TBO, the 7th is the nano 
the5thisthemicrofabricgraftedPB,the6thisthenanofabricgraftedwithTBO,the7thisthenano
fabric grafted with azure A (AA). 
fabricgraftedwithazureA(AA).
Nanomaterials2016,6,243 8of14
3.4. AntifungalActivity—QuantitativeAssessment
The ability of the dyes RB, PB, TBO, and AA immobilized on the fabrics was investigated as
functionsofthephoto-irradiationtime, uptoamaximumof180min. Allthefabricsgraftedwith
RB,PB,AA,andTBOreducedgrowthofallseventypesofpathogensasillustratedbythereduction
oftheODs. ThelargestinhibitionamongthehumanpathogenstestedinthisstudywasonT.viride,
whiletheODreductionofC.globosumandP.funiculosumwasmuchlessthanfortheothers. Theplant
pathogens,M.grisea,displayedthehighestresistancetoalltypesofphotosensitizers,whileP.cinnamomi
showedtheleastresistancetofabricsgraftedwitheachofthedyes. Inordertocompareinhibition
quantitatively among various variables, the four types of photosensitizers and all seven types of
pathogens,weanalyzedtheinhibitionratesandfittedthemusingEquation(4)developedbyKimand
Michielsen[30].
lnOD =lnOD -k [D ]texp(−k t) (4)
t 0min S 0 D
where OD is the optical density of the fungal suspensions at 405 nm. These fungal suspensions
t
wereobtainedbygrowingthefungithatsurvivedaftertminexposuretothetreatedfabricsunder
illumination,asdescribedintheexperimentalsection;k istheinhibitionrateofthegrowth,D isthe
s 0
initialdyeconcentration,tistheexposuretime,andk isthereductionrateofconcentrationofdye
D
byphotobleaching.
Equation(4)wasusedtocomparetheinhibitionrateofeachfabricgraftedwithRB,PB,TBOand
AAagainstsevenpathogensandthephotobleachingrateofthegrafteddyes. Thesignificanceofthe
datawasevaluatedusingthestudentt-testandANOVA.
Thenanofiber-basedfabricgraftedwithRBexhibitedthelargestODreductiononP.cinnamomi
and T. viride with the inhibition rates of 3.7 × 10−2 and 3.5 × 10−2 L/µmol·min, respectively.
M.griseahadthelowestinhibitionrateof1.4×10−2L/µmol·minfortheRB-treatedfabric. Thenext
highestinhibitionrateswereseenwithPB,showing2.5×10−2and2.4×10−2L/µmol·minagainst
P. cinnamomi and T. viride. In general, the inhibition rates for the treated fabrics decreased in
the order RB>PB>TBO>AA. Likewise, the inhibition rates for the fungi decreased in the order
P.cinnamomi>T.viride>A.niger>A.fumigatus>C.globosum>P.funiculosum>M.grisea. Theresults
also show that the inhibition rate for the nanofiber-based fabrics was always higher than for the
microfiber-basedfabrics,asshowninFigure4.
Figure 5 shows the final inhibition percentage and compares the inhibition percentage
of nanofiber-based fabrics with microfiber-based fabrics grafted with photosensitizers.
All nanofiber-based fabrics grafted with photosensitizers showed higher inhibition than the
microfiber-basedfabrics. Theaverageinhibition%innanofiber-basedfabricswasover70%inhibition,
whilemicrofiber-basedfabricsgavelessthan50%inhibition. Finally,theresultsshowedtheincreasing
inhibitionpercentageintheorderofAA<TBO<PB<RBonthegraftedfabrics. Thefabricsgrafted
with AA and TBO showed similar effect but the fabrics grafted with TBO showed slightly higher
inhibitionrates;however,thefabricsgraftedwithRBshowedmuchhigherinhibitionratesthanthe
fabricsgraftedwithPB.Onaverage,theinhibitionrateforthenanofiber-basedfabricswas1.5times
higherthanonthemicrofiber-basedfabricswiththesamephotosensitizer.
Nanomaterials2016,6,243 9of14
Nanomaterials 2016, 6, 243  9 of 13 

FFiigguurree 44.. TThhee oopptitcicaal lddenensistiyt y(O(ODD) r)erdeudcuticotino bnyb nyannaon aondan mdicmroic fraobfraicbsr gicrsafgtreadf twedithw RitBh, RPBB,, TPBBO,T, aBnOd, 
aAnAd gArAapghreadp hase da afusnacftuionnc toiof ntimofet iumnedeurn idlleurmililnuamtiionna toionn (ao)n A(a. )nAig.ern;i g(ber); A(b. )fuAm.ifguamtuigs;a t(uc)s ;T(.c v)iTr.idvei;r i(dde); 
(Cd.) gClo.bgolsoubomsu; (me;) (Pe.) fPu.nfuicnuilcousluomsu;m (f;)( fM)M. g.rgisreiase; aa;nadn d(g()g P).P .cicninnnaammoommi.i .NNootete: :aalll laaxxeess aarree ttoo tthhee ssaammee ssccaallee 
ttoo aaiidd ccoommppaarriissoonnss bbeettwweeeenn mmaatteerriiaallss..  
Figure 5 shows the final inhibition percentage and compares the inhibition percentage of 
nanofiber‐based fabrics with microfiber‐based fabrics grafted with photosensitizers. All nanofiber‐
based fabrics grafted with photosensitizers showed higher inhibition than the microfiber‐based 
fabrics.  The  average  inhibition  %  in  nanofiber‐based  fabrics  was  over  70%  inhibition,  while 
microfiber‐based fabrics gave less than 50% inhibition. Finally, the results showed the increasing 
inhibition percentage in the order of AA < TBO < PB < RB on the grafted fabrics. The fabrics grafted 
with AA and TBO showed similar effect but the fabrics grafted with TBO showed slightly higher
Nanomaterials 2016, 6, 243  10 of 13 
inhibition rates; however, the fabrics grafted with RB showed much higher inhibition rates than the 
fabrics grafted with PB. On average, the inhibition rate for the nanofiber‐based fabrics was 1.5 times 
Nanomaterials2016,6,243 10of14
higher than on the microfiber‐based fabrics with the same photosensitizer.  

FFiigguurree 55.. IInnhhiibbiittiioonn ppeercrceenntataggees sofo tfhteh neannaon aonadn mdimcrioc rfaobfraibcsr iwcsitwh iitmhmimobmiloizbeildiz ReBd, RPBB,, APBA,, AanAd, TaBnOd 
TpBhOotopsheontsoistieznesrist iozne r(sa)o nA.( afu)mAi.gfautmusi;g (abtu) sA;.( bn)igAer.; n(icg)e Tr;. (vci)riTd.e;v (irdi)d eC;.( gdl)oCbo.sgulmob; o(seu)m P;. (feu)nPic.ufluonsuicmul;o (sfu) mP;. 
(cfi)nPn.acminonmaim; aonmdi; (agn) dM(.g g)rMise.ag.r isea.