Concepts and principles of photodynamic therapy as an alternative antifungal discovery platform

16 Pages·2012·1.88 MB·English

by  George P. Tegos

REVIEWARTICLE
published:10April2012
doi:10.3389/fmicb.2012.00120
Concepts and principles of photodynamic therapy as an
alternative antifungal discovery platform
TianhongDai1,2,BethB.Fuchs3,JeffreyJ.Coleman3,RenatoA.Prates4†,ChristosAstrakas5,
TylerG.St.Denis1,6,MarthaS.Ribeiro4,EleftheriosMylonakis3,MichaelR.Hamblin1,2,7 and
GeorgeP.Tegos1,2,8,9*
1WellmanCenterforPhotomedicine,MassachusettsGeneralHospital,Boston,MA,USA
2DepartmentofDermatology,HarvardMedicalSchool,Boston,MA,USA
3DivisionofInfectiousDiseases,MassachusettsGeneralHospital,Boston,MA,USA
4CenterforLasersandApplications,IPEN–CNEN/SP,SãoPaulo,SãoPaulo,Brazil
5DivisionofInternalMedicine,DemocritusUniversityofThraceMedicalSchool,Alexandroupolis,Greece
6DepartmentofChemistry,ColumbiaUniversity,NewYork,NY,USA
7Harvard–MITDivisionofHealthSciencesandTechnology,Cambridge,MA,USA
8CenterforMolecularDiscovery,UniversityofNewMexico,Albuquerque,NM,USA
9DepartmentofPathology,UniversityofNewMexicoSchoolofMedicine,Albuquerque,NM,USA
Editedby: Opportunisticfungalpathogensmaycausesuperficialorseriousinvasiveinfections,espe-
BruceC.Campbell,WesternRegional cially in immunocompromised and debilitated patients. Invasive mycoses represent an
ResearchCentre,USA
exponentially growing threat for human health due to a combination of slow diagnosis
Reviewedby:
and the existence of relatively few classes of available and effective antifungal drugs.
PaulCos,AntwerpUniversity,
Belgium Therefore systemic fungal infections result in high attributable mortality. There is an
SimonAndrewJohnston,University urgent need to pursue and deploy novel and effective alternative antifungal counter-
ofBirmingham,UK measures. Photodynamic therapy (PDT) was established as a successful modality for
*Correspondence: malignancies and age-related macular degeneration but photodynamic inactivation has
GeorgeP.Tegos,Departmentof
only recently been intensively investigated as an alternative antimicrobial discovery and
Pathology,SchoolofMedicine,
CenterforMolecularDiscovery,UNM developmentplatform.Theconceptofphotodynamicinactivationrequiresmicrobialexpo-
HealthSciencesCenter,Universityof sure to either exogenous or endogenous photosensitizer molecules, followed by visible
NewMexico,2325CaminodeSalud, light energy, typically wavelengths in the red/near infrared region that cause the exci-
CRF217AMSC07-4025
tation of the photosensitizers resulting in the production of singlet oxygen and other
Albuquerque,NM87131,USA.
e-mail:[email protected] reactive oxygen species that react with intracellular components, and consequently pro-
†Presentaddress: duce cell inactivation and death. Antifungal PDT is an area of increasing interest, as
RenatoA.Prates,SchoolofDentistry, research is advancing (i) to identify the photochemical and photophysical mechanisms
UniversityNovedeJulho(UNINOVE), involved in photoinactivation; (ii) to develop potent and clinically compatible photosensi-
SãoPaulo,SãoPaulo,Brazil.
tizers; (iii) to understand how photoinactivation is affected by key microbial phenotypic
elements multidrug resistance and efflux, virulence and pathogenesis determinants, and
formation of biofilms; (iv) to explore novel photosensitizer delivery platforms; and (v) to
identify photoinactivation applications beyond the clinical setting such as environmental
disinfectants.
Keywords:photodynamicinactivation,photodynamictherapy,photosensitizer,reactiveoxygenspecies,perme-
abilitybarrier,multidrugeffluxsystems,biofilms,clinicalapplications
INTRODUCTION populations. The diseases include cutaneous, subcutaneous,
FUNGALINFECTIONSANDCOUNTERMEASURES mucosal invasion,and can be found as blood stream infections
Fungi are common causative agents of diseases in both the (BSI)thatarelife-threatening(Gavaldaetal.,2005;Sobel,2007;
immunecompetentaswellastheimmunecompromisedpatient Caston-Osorioetal.,2008;Ameen,2009;Hornetal.,2009;Neofy-
tosetal.,2009;Pappasetal.,2009).Dermatophytosisisprobably
the most prevalent of all fungal diseases but also the least stud-
Abbreviations: ABC,ATP binding cassette; ALA, 5-aminolevulinic acid; APDI,
iedinregardtohost–fungusinteractions(Vermoutetal.,2008).
antimicrobialphotodynamicinactivation;BCVA,best-correctedvisualacuity;BSI,
blood stream infections;CDR1,Candida drug resistance 1;CFU,colony form-
ingunit;CMD,choroidalmajordiameter;CNV,choroidalneovascularization;EPI,
effluxpumpinhibitor;ETC,electrontransportchain;EtNBSe,seleniumanalog
of benzophenothiazinium salt; HOMO,highest occupied molecular orbital; IA, photodynamicinactivation;PDR,pleiotropicdrugresistance;PDT,photodynamic
invasive aspergillosis; IC, invasive candidiasis; IL-1, interleukin-1; LUMO, low- therapy;PEI-ce6,polyethyleneimine-chlorin(e6);PMMA,poly-methylmethacry-
estoccupiedmolecularorbital;MAL,methyl5-aminolevulinicacid;MB,meth- late;PS,Photosensitizer;RB,rosebengal;RD,respiratorydeficiency;RND,resistant
ylene blue; MDR1, multidrug resistance 1; MFS, major facilitator superfam- nodulation(cell)division;RNS,reactivenitrogenspecies;ROS,reactiveoxygen
ily; MIC, minimal inhibitory concentration; NMB, new methylene blue; PDI, species;SOR1,singletoxygenresistance1;TBO,toluidineblue.
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Daietal. Antifungalphotodynamictherapy
Fungal diseases are endemic in certain parts of the world and triazoles that are notorious for causing drug–drug interactions
include blastomycosis, chromoblastomycosis, coccidioidomyco- andtoxicity;(2)theformulationofamphotericinBwaschanged
sis,histoplasmosis,paracoccidioidomycosis,penicilliosis,orpan- toachievebetterabsorptionasthisdrugisnowavailableasalipid
demic including invasive aspergillosis (IA), invasive candidiasis formulationencapsulation.Thismodificationhasreducedtoxic-
(IC),cryptococcosis,dermatophytosis,fusariosis,andmucormy- ityduetooff-targeteffectsonhostcellsbuthasanincreasedcost;
cosis. Very likely, the greatest threat to life results from those (3) enhanced activity has been observed with two of the newer
pathogens that cause BSI, yet with IA and IC diagnosis is not triazoles,posaconazole,andvoriconazole;(4)newtriazolesarein
easyandoftenthesediseasesaretreatedempiricallywhenblood development(albaconazoleandravuconazole);(5)echinocandins
cultures are negative for bacterial pathogens. In the case of IC, are slow in development and ineffective against C. neoformans.
therecanbenumerousriskfactorsthat,forthemostpart,arenon- While β-1,3-glucan is present in the cell wall of this pathogen,
specific.Candidaspp.arethethirdleadingcauseandareassociated caspofungin may have reduced activity against the β-1,3-glucan
withthehighestmortalityof catheter-relatedinfections(Crump synthase(Feldmesseretal.,2000);(6)drugresistancetotriazoles
andCollignon,2000).AlthoughpreventionofICusingazolepro- is a common feature of several species of Candida; an increase
phylaxiscanbeeffectiveinselectedhigh-riskpatientpopulations, inresistancetoechinocandinsisfrequentlyreported;and,impor-
selectionforinvasiveinfectionbyresistantnon-albicansCandida tantly,(7)theimpressivenumberofreferencestoresistancedevel-
speciesormoldsisapotentiallydevastatingconsequence.Despite opingtoantifungals(Pfalleretal.,2010)makesthedevelopmentof
improvementsinantifungaltherapy,themortalityrateduetoCan- newantifungalcountermeasuresanecessity.Onepromisinganti-
didaBSIhasimprovedlittleoverthelasttwodecades,remaining fungal modality is the light-based technology of photodynamic
highat15–49%(Gudlaugssonetal.,2003).ABSIepisodesignifi- therapy(PDT).Thisreviewsummarizestheprogressinantifungal
cantlyincreasescostofcare.Inoneanalysis,theestimatedcostof PDTattemptingtoformulatetheprinciplesaswellastheconcepts
anICBSIepisodewas$34,123perMedicarepatientand$44,536 foritssuccessfulimplementation.
per private insurance patient (1997, US$), with an overall eco-
nomic burden of $2 billion dollars annually in the USA (Rentz THEPLATFORMOFPHOTODYNAMICTHERAPY
etal.,1998). Photodynamic therapy (St. Denis et al., 2011a) was discovered
Theincreasedinfectionofimmunocompromisedhostsisdra- in1900byOskarRaabandHermannvonTappeinerwhofound
maticallyillustratedbyCryptococcusneoformans,apathogenthat that Paramecium spp. protozoans were killed after staining with
rosetoprominenceasthecausativeagentofcryptococcosis,which acridine orange and subsequent exposure to bright light (Raab,
isalife-threateningdiseasethathasemergedinparallelwiththe 1900).Inthe1970s,PDTwasinitiallydevelopedasatherapyfor
HIV/AIDS epidemic. Cryptococcosis results from inhalation of cancer after it was discovered that porphyrins selectively local-
fungal cells with subsequent lung infection and pneumonia. In izedintumors(MittonandAckroyd,2008).Sincethen,PDThas
theabsenceofaneffectiveimmuneresponse,thefunguscandis- been clinically used for treatment of various malignancies and
seminatetothebraintocausemeningoencephalitis,thesymptoms is an approved therapy for destruction of choroidal neovascu-
ofwhichincludeheadache,fever,visualproblems,andanaltered larization (CNV) in age-related macular degeneration. Recently,
mentalstate(BrizendineandPappas,2010).C.neoformanscauses antimicrobialPDThasbeenproposedasanalternativeapproach
an estimated one million cases of meningoencephalitis globally forlocalizedinfections(St.Denisetal.,2011a).
peryearinpatientswithAIDS,leadingtoapproximately625,000 Photodynamic therapy involves the use of a non-toxic light-
deaths(Parketal.,2009).Thebulkofthisdiseaseincidenceisin sensitivedyecalledaphotosensitizer(PS)combinedwithharmless
sub-SaharanAfrica,wherefatalcasesofcryptococcosismayexceed visiblelightof theappropriatewavelengthtomatchtheabsorp-
deaths from tuberculosis in some areas (Park et al., 2009). The tionspectrumofthePS.AfterphotonabsorptionthePSreaches
importance of immune suppression in predisposition to infec- anexcitedstatethatcanundergoreactionwithambientoxygen,
tion has been challenged by the recent discovery of specialized resultingintheformationofreactiveoxygenspecies(ROS).PDT
interactionsbetweenthefungusanditsmammalianhosts,andby isahighlyselectivemodalityas(i)thePScanbetargetedtothe
theemergenceof therelatedspeciesCryptococcusgattii asapri- unwanted cells or tissue (Hunt,2002) and (ii) cell death is spa-
marypathogenofimmunocompetentpopulations(D’Souzaetal., tiallylimitedtoregionswherelightoftheappropriatewavelength
2011).Asetofrecentlydiscoveredcryptococcalpathogenesisfea- isapplied.SincesomePSbindrapidlyandselectivelytomicrobial
turesrevealthefungaladaptationtothemammalianenvironment cells,itwassuggestedthatPDTcouldbeusedasananti-infective
(Alanioetal.,2011).Thesefeaturesincludenotonlyremarkably approach; this became a reality in the mid 1990s (Nitzan et al.,
sophisticatedinteractionswithphagocyticcellstopromoteintra- 1992).
cellularsurvival,disseminationtothecentralnervoussystem,and
escape(Alanioetal.,2011;Kronstadetal.,2011),butalsosurpris- THEPHOTOPHYSICALPROCESSESOFPDT
ingmorphologicalandgenomicadaptationssuchastheformation ThethreeprinciplecomponentsofPDTarethePS,visiblelight,
ofpolyploidgiantcellsinthelung(Fuchsetal.,2010). andoxygen.Theseindividuallyharmlesscomponents,whencom-
Thestateoftheartinantifungaldrugdiscoveryincludesonly bined,yieldpotentROS(Figure1).Dyeshavemolecularstructures
a few new compounds which offer some hope for eradication thataretypifiedbyconjugateddoublebondscontainingadelocal-
of fungal diseases (Ostrosky-Zeichner et al., 2010). Specifically: izedsystemofπ-electrons.InthePSground(singlet)state,these
(1) the echinocandins act against a specific component of fun- electrons are spin paired in low energy orbitals. Upon applica-
gal pathogens. As such,their safety profile is quite good,unlike tionoflightcorrespondingtotheabsorptionpeakofthePS,the
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Daietal. Antifungalphotodynamictherapy
O ,yieldingsuperoxideanionO•−)whichcanthengoontoform
2 2
other ROS including the hydroxyl radical (•OH),and hydrogen
peroxide(H O ).
2 2
The ROS formed through the Type 1 process have a range
•
ofdifferentreactivities(Ochsner,1997). OH,arguablythemost
reactiveof thethreeROSformed,isastrongelectrophilethatis
abletochemicallyattackaverywiderangeofbiomolecules.H O
2 2
•− •−
islessreactiveandO isleastreactive.Nonetheless,O maybe
2 2
convertedtoH O andO bysuperoxidedismutase.H O isonly
2 2 2 2 2
consideredtrulyreactivewhenitreactswithferrousironinwhat
isknownastheFentonreaction.
H O +Fe2+ →OH- +•OH+Fe3+
2 2
whichresultsinthehomolyticfissionoftheoxygen–oxygenbond
FIGURE1|Schematicillustrationofphotodynamictherapyincluding •
in H O to yield a hydroxide ion and OH via the oxidation of
theJablonskidiagram.ThePSinitiallyabsorbsaphotonthatexcitesitto 2 2
ferrousirontoferriciron(Valkoetal.,2005).H O isremoved
thefirstexcitedsingletstateandthiscanrelaxtothemorelonglivedtriplet 2 2
•
state.ThistripletPScaninteractwithmolecularoxygenintwopathways, throughcatalase,formingwater,andoxygengas.Although OH
typeIandtypeII,leadingtotheformationofreactiveoxygenspecies(ROS) isnotbrokendownbyanenzymaticreaction,itmaybequenched
andsingletoxygenrespectively. byantioxidants,includingantioxidantpeptides(e.g.,glutathione)
orbyantioxidantvitamins(e.g.,ascorbicacid).
Because1O isnotaradical,itreactswithbiologicalmolecules
electroninthehighestoccupiedmolecularorbital(HOMO)ofthe 2
through quite different mechanisms, making the Type II path-
PSisexcitedtothelowestunoccupiedmolecularorbital(LUMO),
wayresponsiblefordifferentmacromolecularreactionpathways.
excitingthePStoanunstable,andshortlivedexcitedsingletstate.
1O tendstofavorreactingwithdoublebondsandsulfurmoieties
In this state, several processes may rapidly occur (Foote, 1991). 2
(bothofwhichhavehighelectrondensities)andmayinteractwith
ThemostimportantofthesetothePDTprocessisthereversalof
aromaticcomponentsofmacromoleculesinDiels–Aldercycload-
theexcitedelectron’sspin,knownasintersystemcrossingtogive
ditions (Leach and Houk, 2002; Singleton et al., 2003), 1O is
thetripletstateofthePS.Thistripletstateislessenergeticthanthe 2
unable to be broken down by enzymes but can be quenched by
excitedsingletstate,buthasamuchlongerlifetime(microseconds
antioxidants.
asopposedtonanoseconds),astheexcitedelectron,nowwitha
spinparalleltoitsformerpairedelectron,maynotimmediately PROPERTIESOFPHOTOSENSITIZERS
revert to a lower energy level according to the Pauli Exclusion Photosensitizerareusuallyorganicdelocalizedaromaticmolecules
Principle.Accordingly,theexcitedelectroninthePStripletstate consistingoflargeconjugatedsystemsofdoublebondsthatmay
may change its spin orientation (a relatively slow process) and beconsideredasacentralchromophorewithauxiliarysidechains
emititsenergyasphosphorescence,oralternativelythetripletPS attached (auxochromes) which are responsible for further elec-
mayinteractwithmoleculesabundantinitsimmediateenviron- trondelocalizationofthePS,thusalteringtheabsorptionspectra
ment.Becauseoftheselectionrulesthatspecifythattriplet–triplet of the PS (Wainwright et al., 2006). Due to extensive electron
interactionsarespin-allowedwhiletriplet–singletinteractionsare delocalization, PS tend to be deeply colored. This means that
spin-forbidden,thePStripletcanreactreadilywithmolecularoxy- theenergyrequiredtoexcitetheelectronsintheHOMOtothe
genthatisoneofthefewmoleculesthatareatripletintheground LUMOislowcomparedtolessdelocalizedmoleculesandtherefore
state(Figure1). theabsorptionbandsareinthelongerwavelength(red)spectral
regionandarelarge,reflectingthehighprobabilityofexcitation.
THEPHOTOCHEMICALGENERATIONOFOXIDIZINGSPECIES Acridine orange was the first photodynamic agent used (Taka-
TheabilityofPDTtoproduceROSneedsthepresenceofmolecu- hashi et al.,1975). Most of the PS that have been employed for
laroxygen(O ;Foote,1991;Tanielianetal.,2000).Asmentioned thetreatmentofcancerandothertissuediseasesarebasedonthe
2
above,thegroundelectronicstateofoxygenisatriplet,whereby tetrapyrrolenucleus,whichwereinitiallyporphyrins.Duetothe
thetwooutermostorbitalsareunpairedbutspinparallelandthis unfavorableabsorptionspectrumofporphyrins(lowpeaksinthe
tripletcanundergoenergytransferuponcollisionwiththeexcited 630-nmregion),emphasisthenshiftedtoothertetrapyrrolessuch
PStriplet.ThisTypeIIprocessinvolves“flippingthespin”ofthe aschlorins,bacteriochlorins,andphthalocyaninesthathavemuch
outermostO electronandshiftingitintotheorbitalcontaining largerpeaksatlongerredwavelengthswheretissuetransmission
2
theotherelectron,whichinturnleavesoneorbitalentirelyunoc- oflightismaximal.Whilethesetetrapyrroleswereinitiallystudied
cupied(aviolationofHund’srule).Termedsingletoxygen(1O ), asantimicrobialPS,theirinabilitytokillpathogensthatbelonged
2
thisformof oxygen(notconsideredaradicalasitselectronsare to other classes than Gram-positive bacteria led to the proposal
spin paired) is extremely short lived and reactive, owing to its ofdyeswithdifferentmolecularframeworksasantimicrobialPS
electron configuration instability. An alternative photochemical (Sharma et al.,2011). Phenothiazinium salts,such as methylene
mechanismistermedtheTypeIwhenthePStripletdirectlytrans- blue(MB)andnewmethyleneblue(NMB;Figure2A,Brespec-
fers an electron,sometimes in concert with proton donation to tively;Harrisetal.,2005;Souzaetal.,2010)areoftenusedsince
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Daietal. Antifungalphotodynamictherapy
FIGURE2|RepresentativechemicalstructuresofPSthathavebeen (H).Conjugatebetweenpolyethylenimineandchlorine(e6),PEI-ce6(Tegos
reportedtobeespeciallyactiveinthephotoinactivationoffungal etal.,2006);(I),BF2ChelateofN-(4-(4-Bromo-2-
cells.(A)Methyleneblue,MB,(B),newmethyleneblue,NMB(Daietal., (4-bromo-3-(4-((diethyl(methyl)-ammonio)methyl)phenyl)-5-(4-
2011a);(C),seleniumNileblueanalog,EtNBSe(Foleyetal.,2006);(D), methoxyphenyl)-1H-pyrrol-2-ylimino)-5-(4-methoxyphenyl)-2H-pyrrol-3-
malachitegreenoxalate,(E),bis-amino-substitutedbacteriochlorin,BC29 yl)benzyl)-N-ethyl-N-methylethanaminiumiodide(Frimannssonetal.,
(Huangetal.,2010a);(F),tris-N-methyl-pyrrolidiniumfullerene,BB6(Tegos 2010);(J),1,3-bis(dimethylamino)-2-propoxy-methoxysilicon
etal.,2005);(G),hexakis-cationicfullereneBB24(Huangetal.,2010b); phthalocyanine,BAM-SiPc(Soetal.,2010).
theywerethefirstantimicrobialPStobetestedandareclinically polycationic(PS)conjugatesbetweenpolyethyleneimineandchlo-
approvedforhumanuse.Althoughtherehasbeenawiderange rin(e6), PEI-ce6, (Figure 2H; Tegos et al., 2006), tris-cationic
of antimicrobial PS reported,it is becoming clear that the opti- substitutedfullerenes(BB6;Figure2F),hexakis-cationicfullerenes
mal structures for inactivating bacterial cells and the optimum (BB24;Figure2G),thecationicporphyrinTriP[4],(Lambrechts
structure for fungal cells may in fact be subtly different from etal.,2005),andtheseleniumanalogof benzophenothiazinium
those used for cancer. Some structures such as protease-stable salt(EtNBSe;Figure2C)maybeequallypotenttomediatephoto
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Daietal. Antifungalphotodynamictherapy
killing of bacteria and fungi. However other structures, includ- PS to eradicate a microbial population the influence of PDI on
ingPSthatareusuallyconsideredtobespecificforkillingcancer thebarrierpropertiesoftheplasmamembranewasstudied.TBO
cells,havealsobeenreportedtobeeffectiveinkillingfungalcells mediated PDI-induced a permeability change proceeding in an
(mainlyC.albicans).ThisappliestoPSsuchasPhotofrin(Bliss “all-or-none”fashionwhichwasreflectedinpotassiumionsand
et al., 2004), Al(III)-tetrasulfonated phthalocyanine (Bertoloni E260-absorbing material efflux, reduction of the cell volume,
et al., 1992; Lazarova, 1993; Bliss et al., 2004), and PC4 silicon and vacuole integrity. Finally, it was observed that the loss of
phthalocyanine (Lam et al., 2011). Moreover some PS that are cell viability was not induced by the all-or-none loss of barrier
highlyeffectiveinkillingGram-positiveandGram-negativebac- properties(Paardekooperetal.,1992).Arecentstudyclaimsthat
teria are not very effective in killing fungal cells. It appears that MB-mediatedPDIincreasesmembranepermeabilityinC.albicans
having a large number of cationic charges is important for effi- (Giroldoetal.,2009).
cientphotodynamicinactivation(PDI)ofGram-negativebacteria ThecombinationoftheanionicPSrosebengal(RB)withglu-
andtoalesserextentGram-positivebacteria,butthisstructural tathione was effective in killing C. albicans. Although the key
featuredoesnotprovidegoodbindingorpenetrationintofungal hypothesis was enhancement of PDI involving stimulation of
cells. Rather,more lipophilic structures with a lower amount of the singlet oxygen generation, the effect of glutathione on the
cationicchargeseemtobebetterforfungalcells.Anexampleof permeabilitybarrierofeukaryoticcellsiswellestablished.Alterna-
thislatterclassisthebacteriochlorin(BC29;Figure2E)thatwas tively,compoundsthatcreateporeswithinthefungalmembrane
effectiveatkillingCandidacells,whileanalogswithmorecationic have shown increased influx of PS compounds that are other-
chargeswerebetteratkillingbacteria(Huangetal.,2010a).The wise unable to enter the cytosol. Phytoalexins of the saponin
listofPStargetingexclusivelyC.albicansisexpandingandinclude family are able to bind to the fungal sterol ergosterol, creating
malachitegreen(Figure2D;Souzaetal.,2010)anunsymmetrical poresinthemembraneresultingincellularleakage(Simonsetal.,
bis-aminophthalocyaninebis-aminosilicon(IV)phthalocyanine 2006). Pretreatment of C. albicans cells with a sub inhibitory
(BAM-SiPc;Figure2J;Soetal.,2010).Abrominatedborondiflu- concentration of a saponin significantly increased the uptake of
oride(BF )chelatedtetraarylazadipyrromethenephotosensitizer the PS molecules RB and chlorin (e6),and was able to decrease
2
(Figure 2I; Frimannsson et al., 2010) reduced in combination the survival fraction after exposure with the correct wavelength
with light the viability of yeast cells [5.7 log (10)]. Photodit- of a low-intensity light (Coleman et al., 2010). The degree of
hazine,aglucosaminesaltofchlorine6,enhancedtheinactivation C. albicans killing was 2–5logs greater in the presence of the
of C. guilliermondii cells by visible light (Strakhovskaia et al., saponin when compared to the PS molecules alone. Pretreat-
2002). ment of the saponin did not result in any significant increase
As microorganisms produce and accumulate porphyrins,the in C. albicans killing when PEI-ce6 was used as the PS, pre-
appealinghypothesisofendogenousphotosensitizationisalsoan sumably because of the high permeability of the conjugated PS
alternativepathwayofphotoinactivation(OrielandNitzan,2010). molecule.Confocalmicroscopyrevealedthatce6resultedinmin-
In this approach a small non-dye aminoacid (5-aminolevulinic imalcellularuptakeinC.albicans,howeverco-incubationofthe
acid, ALA) is administered as it has been shown that exoge- PS and the saponin for 1h resulted in a visible increase of PS
nously supplied ALA enters into the microbial heme biosyn- withintheC.albicans cytosol(Figure3).Collectively,thisstudy
thetic pathway and results in an accumulation of excess pro- suggests in addition to saponins, other compounds with fun-
toporphyrin IX than can act as a moderately effective PS. In gal pore-forming properties could be used in conjunction with
an alternative approach, extracts from Alternanthera maritima a PS molecule,and compounds with antifungal efficacy derived
(seaside joyweed) with an absorption range at 650–700nm can by pore-forming ability such as the polyenes (amphotericin B
effectively photoinactivate Candida dubliniensis. The chemical and nystatin) may be a potent treatment strategy for fungal
compositions of the extracts were determined by chromato- infections.
graphic and spectroscopic techniques. The results suggest inhi-
bition of the growth of C. dubliniensis after being irradiated
with a 685-nm diode laser irradiation alone or crude extracts
at 25mg/ml did not significantly reduce the number of colony
forming units (CFU) per milliliter. Steroids, triterpenes, and
flavonoids were identified in the analyzed extracts (Gasparetto
etal.,2010).
BYPASSINGTHEPERMEABILITYBARRIER
Bypassing the permeability barrier is a common and challeng-
ing theme in antimicrobial drug discovery. Yeasts and fungal
pathogens are variable in their cell envelopes, possessing outer
wallmixturesofglucans,mannan,andchitinpolymers.Thisfea-
turemakestheminherentlymorepermeabletoexternalsubstances FIGURE3|ConfocallaserscanningmicroscopyofC.albicanscells
thanGram-negativebacteria.Inoneofthefirstreportsforantifun- treatedwithce6after24h.Leftpanel–Yeastcellstreatedwith100μM
ce6alone.Rightpanel–Yeastcellstreatedwithce6and4μg/mlofa
galPDI,Toluidineblue(TBO)wastestedagainstKluyveromyces
saponin.Scalebarrepresents20μm.
marxianus (Paardekooperetal.,1992).Apartfromabilityof the
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Daietal. Antifungalphotodynamictherapy
PDIFORAZOLEANDANTIFUNGALRESISTANTPHENOTYPES
ThereisdocumentedevidencethatPDIisequipotentagainstboth
antimicrobial-resistantandsusceptiblemicroorganisms(St.Denis
etal.,2011a).Thismaybeobservedinazole-resistantandsuscep-
tibleCandidaspp.PDIemployingPhotogem®asaPSwithalight
emittingdiodehadasignificanteffectagainstfluconazole-resistant
C.albicans andCandidaglabrata (Dovigoetal.,2011a).Asetof
clinicalisolatesfromadultHIVpatients,includingstrainshighly
resistant and sensitive to fluconazole and amphotericin B, were
subjectedtoPhotofrin®mediatedPDIusinga630nmlaserand
theappropriatetimetodeliver45–135J/cm2.Therewasnotsub- FIGURE4|MicroscopywasusedtovisualizelocationofthePEI-ce6in
stantialdifferenceinthesurvivalfractionsbetweenresistantand associationwithC.neoformans.Cellsweregrownat37˚C,exposedto
sensitiveisolates(Mangetal.,2010). PEI-ce6for10minandthenwashedwithPBS.Arrowsindicatelocalization
ofthePEI-ce6atthecellwallperimeter.Arrowheadsmarkthelocalization
However,earlystationaryphaseyeastformsofC.albicansand
ofPEI-ce6withintheinteriorofthecell.Withwild-typecells(KN99α),the
C.glabratawerenotadverselyaffectedbytreatment.Respiratory- dyewasconfinedtothesurfaceofthecells.However,withtheKN99α
deficient (RD) strains of C. albicans and C. glabrata display a rom2mutant,thedyewasabletopenetratethecellsurface.
pleiotropic resistance pattern,including resistance to azole anti-
fungals,thesalivaryantimicrobialpeptideshistatins,andcertain
typesoftoxicstresses.ThecationicporphyrinPSmeso-tetra(N- (TEM). Cell wall defects are enhanced at the high temperature
methyl-4-pyridyl)porphinetetratosylate(TMP-1363)iseffective growthcondition37˚C,astressconditionforthecell(Fuchsetal.,
inPDTagainstyeastformsofC.albicansandC.glabrata.Incon- 2007a).Thelackofastablecellwallstructureislikelyassociated
trasttothispattern,RDmutantsofbothC.albicansandC.glabrata withtheactinandmicrotubuledefects,cytoskeletalcomponents
were significantly more sensitive to PDI compared to parental needed to transport structural components to the cell wall and
strains.Thesedatasuggestthatintactmitochondrialfunctionmay maintain a rigid configuration and uniform shape. The result
provideabasallevelof antioxidantdefenseagainstPDI-induced of theculminationof defectswasanincreaseincelldeathupon
phototoxicityinCandidaspp.,andrevealspathwaysofresistance applicationoftheactivatedPS.
tooxidativestressthatcanpotentiallybetargetedtoincreasethe Interestingly, cell death was enhanced with the addition of
antifungal efficacy of PDT (Chabrier-Roselló et al.,2008). Cur- theantifungalcaspofungin.Whilecaspofunginiseffectiveagainst
cumin,anaturallyoccurringpigmentwithamaximumabsorption other fungal pathogens, including C. albicans, it fails to exhibit
peakintheshortwavelengthrangebetween408and430nmhas fungicidaleffectsagainstC.neoformans,forreasonsthathavenot
beenrecentlyusedforsuccessfulPDIinclinicalisolatesofCandida yet been fully elucidated (Abruzzo et al., 1997; Espinel-Ingroff,
spp.(Dovigoetal.,2011b). 1998).Asamemberoftheechinocandinclassofdrugs,caspofun-
gintargetstheglucansynthaseFks1p.Inthiscase,theinclusionof
INACTIVATIONOFCRYPTOCOCCUS ANDOTHERFUNGAL caspofunginenhancedthekillingoftheactivatedPS.Eventhough
PATHOGENS itdoesnotkillC.neoformans,caspofunginisknowntoaffectthe
RecentstudieshaverevealedasubstantialPDTeffectbothinCryp- β-glucan linkages (Feldmesser et al., 2000). It is suspected that
tococcusspp.aswellasavarietyoffungalpathogens.Theseinclude thecombinationof thetwocellwalltargetedcompoundsdually
bothmechanisticandconceptualPDIstudies.Stresssensorsdetect damagedthecellenoughtoleadtocelldeathorthatcaspofungin
ROS and the cell wall elicits a response noted by the activation weakenedthecelltoallowforgreaterpenetrationofthePSthatled
of Slt2 for Saccharomyces cerevisiae or Mpk1 for C. neoformans tocelldeath.TheresultssuggestthatexternalsourcesofROScan
(Vilellaetal.,2005;Geriketal.,2008).InC.neoformans PKC1is beappliedasameanstoinhibitfungi.Potentially,itcouldenhance
essentialfordefenseagainstoxidativestress,bothintheformof thefungicidalactivityofcompoundsthattargetorweakenthecell
hydrogenperoxideandthethioloxidizingagent,diamide(Gerik wall.
etal.,2008). Asecondstudytestedtheeffectivenessof TBOemployingC.
FortheC.neoformans cellwalldefectivestrainKN99αrom2, gattii strains with distinct susceptibility profile to amphotericin
the PS PEI-ce6 exhibited an increased association with KN99α Bandazoles.TheconceptofequipotentPDIbetweensusceptible
rom2,whichwasobservedtopenetratethecell(Fuchsetal.,2007b; andresistantisolateswasre-confirmed.Amoredetailedmecha-
Figure 4). Rom2 is a guanyl nucleotide exchange factor in the nisticstudywasattemptedincludingdeterminationof ROSand
cellwallintegritypathway,relayingtheactivationof thesensors reactivenitrogenspecies(RNS,e.g.,peroxynitrite)productionand
byextracellularstressortothesignalingcascadethatinitiatesthe thecatalaseandperoxidaseactivitiesweremeasured(Soaresetal.,
phosphorylation events of the mitogen activated protein kinase 2011).
(MAPK)cascade(Ozakietal.,1996;PhilipandLevin,2001).The AvarietyofreportstestedtheuseofdifferentPSandexplored
association and ability of the PS to penetrate and collect within optimalconditionsandthemorphologicalchangesobservedupon
the cell was likely due to defects in the cell that prevent the PS PDIofthedermatophyteTrichophytonrubrum(Kampetal.,2005;
frombeingexcluded.Therom2mutantischaracterizedbya35% Smijsetal.,2007,2008;SmijsandPavel,2011).Thelistof PDI-
reduction in the amount of β-glucan and physical disparities in inactivatedfungalspecieshasbeenincreasingexponentially.Tri-
thecellwallobservablethroughtransmissionelectronmicroscopy chophyton mentagrophytes,Trichophyton tonsurans,Microsporum
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Daietal. Antifungalphotodynamictherapy
cookei,Microsporumgypseum,Microsporumcanis,Epidermophyton increase of phototoxicity when compared with the PDI efficacy
floccosum, Nannizia cajetani, Metarhizium anisopliae,Aspergillus of MB in the absence of EPIs. In addition, pre exposure of the
nidulans,A.fumigatus,andFusariumsp.,havebeentestedwitha CDR1/CDR2 overexpressing mutant YEM 15 in verapamil (+)
varietyofdifferentPS,offeringanewavenueforantifungalthera- andsubsequentAPDIrevealed2logsofcellreductioninthepres-
pies(Friedbergetal.,2001;Calzavara-Pintonetal.,2005;Gonzales ence of the EPI, in comparison with virtually no effect in the
etal.,2010). absenceoftheABCpumpblocker.Theauthorsdemonstratedthat
ABC pumps are directly implicated in MB efflux from the cell
FUNGALEFFLUXSYSTEMSASAPDITARGET cytoplasm.BoththeinfluxandtheeffluxofMBmayberegulated
There are two main types of fungal drug efflux systems (Can- by MFS systems and blocking this gate before incubation with
non et al., 2009). (1) The major facilitator superfamily (MFS) MBcandecreasetheuptakeandAPDIeffects.AnABCinhibitor
transporters use the electrochemical gradient across the plasma couldbeusefullycombinedwithMB-APDTfortreatingC.albicans
membranetoexpeldrugsfromcells,and(2)ATPbindingcassette infections.
(ABC) transporters, in contrast, use ATP binding and hydroly- This interaction seems to be less obvious for different PS
sistoeffluxdrugs.Althoughfungalcellscontainmanygenesfor chemotypes. The participation of efflux systems in porphyrin
bothtypesofsystems,clinicalazoleresistanceismostoftenasso- mediatedPDIhasbeenimpliedinmammalianABCtransporter
ciated with overexpression of ABC transporters (Holmes et al., systems(Morganetal.,2010).
2008; Cannon et al., 2009). There are several classes of fungal
ABCtransporters,andthepleiotropicdrugresistance(PDR)fam- BIOFILMERADICATION
ilyisoftenresponsibleforfungaldrugresistance(Lampingetal., Microorganismsinnaturethrivethroughadherencetobothliv-
2010).ClinicallyimportantPDRtransportersincludeC.albicans ingandinanimatesurfacesviabiofilmformation(Pflumm,2011).
Cdr1p(CaCdr1p)andCaCdr2p,whicharehomologsoftheSac- The dense and protected environment of the biofilm,as well as
charomyces cerevisiae Pdr5p (ScPdr5p) and mammalian G-type the significantly different phenotypic properties of biofilm cells
ABCtransporters(Holmesetal.,2006,2008;Cannonetal.,2009). fromfree-floatingcellsofthesamespecies,havebeenimplicated
FungalPDReffluxsystemshaverelativelypromiscuoussubstrate in giving rise to as much as 1000-fold resistance to antimicro-
specificitiesthatarepresumablydefinedbytheirtransmembrane bials (Lewis, 2001). Biofilm formation is a critical event in the
domains. These specificities often partially overlap among fam- developmentofcandidosis,includingdenturestomatitis(chronic
ily members in a particular organism and thus provide broad- atrophic candidosis),which can affect up to 65% of edentulous
spectrum protection against xenobiotic threats, including those individuals (Chandra et al., 2001). Despite the use of antifun-
posed by the widely used and well-tolerated azole and triazole galdrugstotreatdenturestomatitis,infectioncanoftenbecome
drugs. re-established. By using a [poly(methyl methacrylate), PMMA]
The role of multidrug efflux in antimicrobial PDT resistance biofilmmodel,itwasdemonstratedthatC.albicans biofilmsare
has only recently come under scientific investigation. Phenoth- potentiallyhighlyresistanttothecurrentlyusedantifungalagents.
iaziniumdyesMBandTBOareamphipathiccationsandphysic- Drugresistancewasalsoshowntodevelopwithtimeandcorre-
ochemicallysimilartotheantibacterialalkaloidberberine,awell- latedwithbiofilmmaturation(Chandraetal.,2001).Thereisan
characterized substrate of MFS efflux systems in Gram-positive expandingbodyof literatureregardingPDT-basedbiofilmerad-
bacteria (Tegos et al., 2002). This similarity raised the possibil- ication strategies, with emphasis on the use of different PS for
itythatphenothiaziniumPScouldalsobesubstratesofmicrobial biofilm related phenotypes and microbial species (Biel, 2010).
effluxsystems.Recentexperimentalevidenceindicatedthatphe- The eradication of microbial biofilms remains a key challenge
nothiaziniumswereNorA(MFS)substratesinS.aureusandpossi- in the antifungal discovery agenda and new efforts are required
blyMexABresistancenodulation(cell)division(RND)substrates toaddressanumberofclinicalconditions.Thelistincludesuri-
inP.aeruginosa(TegosandHamblin,2006).Theobservationthat nary tract infections, catheter infections, middle-ear infections,
ABC transporters and not MFS affect MB-mediated antimicro- formationof dentalplaque(Fontanaetal.,2009),periodontitis,
bialPDI(APDI)inthepathogenicyeastC.albicansisperplexing (Raghavendraetal.,2009)gingivitis,endodontics,(Soukosetal.,
(TegosandHamblin,2006;Pratesetal.,2011).Pratesetal.tested 2006)osteomyelitis(Bislandetal.,2006),infectedcontactlenses,
this hypothesis by comparing MB-PDI for two pairs of isogenic endocarditis,andinfectionsofpermanentindwellingdevicessuch
C.albicansstrains(i)YEM12andYEM13mutantoverexpressing as joint prostheses heart valves and implants (Schuckert et al.,
MDR1MFS;and(ii)YEM14andYEM15mutantoverexpressing 2006).
CDR1/CDR2(Figure5).SinceeffluxsystemsaffectAPDIofMBin There is a wealth of literature describing PDT-based anti-
C.albicans,alogicalstrategywastoexplorewhethereffluxpump biofilm strategies that focuses mostly on the use of different
inhibitors(EPIs)couldpotentiateAPDIwithMB.Theyusedtwo PS against a variety of microbial species (Biel, 2010). In con-
well-documentedEPIs,oneforeachcorrespondingeffluxsystem, trast there are only a limited number of studies exploring the
INF (targetingMFS)andverapamil(+;targetingABCsbothin effects of PDT on phenotypic biofilm elements (e.g.,adhesions;
271
yeastandmammaliansystems)andthereferenceC.albicansstrain Soaresetal.,2008).Moreover,thereisnoconsensusastowhich
DAY185.PreexposuretoINF followedbyMB-mediatedPDI, is the most reliable model for evaluating PDT efficacy against
271
resultedinaparadoxicalprotectionratherthanenhancementof biofilms. The majority of published reports use methodologies
phototoxicity.Ontheotherhand,preexposureofC.albicanscells wherebiofilmsaregrownin/onplasticorsiliconmicrotiterplates
to verapamil (+) and subsequent MB-mediatedAPDI led to an andsurfaces.Thesebioassayshavebeenrepeatedlycriticizedfor
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Daietal. Antifungalphotodynamictherapy
FIGURE5|Effectofeffluxpumpsonphotodynamicinactivation 488nm.Wepresentthreepicturesofthesamefield.Red
ofC.albicans.ConfocalmicroscopyimageofYEMstrains.Cells correspondstothefluorescenceofMBandgreencorrespondstothe
wereincubatedwith10μMR123and100μMMBandexcitedat fluorescenceofR123.
lack of robustness and occasionally yield inconsistent results. In 2011).Inamuchmorechallengingscenario,thecombinationof
this context it has been demonstrated that C. alibcans biofilms gasiformozoneandMB-mediatedPDIwereunabletoreducethe
are sensitive to Photofrin® PDT (Chabrier-Rosello et al.,2005), viablecountsonamultispeciesmatureoralbiofilmassembledby
C. albicans and C. dubliniensis were susceptible to erythrosine- Actinomyces naeslundii,Veillonella dispar, Fusobacterium nuclea-
mediatedPDT,butthebiofilmsofbothCandida spp.weremore tum,Streptococcussobrinus,S.oralis,andC.albicans(Mülleretal.,
resistantthantheirplanktoniccounterparts(Costaetal.,2012). 2007).
The combination of erythrosine- and RB-mediated PDT have Thebulkof cellsinbiofilmsareactuallyhighlysusceptibleto
some effect in biofilms but also was effective in reducing and killingbyantimicrobialsanditisindeedonlyasmallfractionof
destroyingof C.albicans blastoconidiaandhyphae(Costaetal., cellsknownaspersistersthatremainalivefollowingantimicrobial
2012).MB-PDTcombinedwithInGaAlPlaser(660nm)exhibited treatments(Lewis,2010).Persistersrepresentasubpopulationof
amodestreductioninbiofilm C.albicans species(Pereiraetal., cells that spontaneously go into a dormant, non-dividing state.
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Daietal. Antifungalphotodynamictherapy
When a population is treated with a fungicidal antibiotic, reg- resistanceinthesethreemajorhumanpathogensafter20dailypas-
ular cells die but the persisters survive. This persister pheno- sageswasstudied.Simultaneouslyforthesamestrains,theability
type hypothesis has been proven for C. albicans biofilms using of daily sequential subcultures in sub inhibitory concentrations
amphotericin B and chlorhexidine (Lafleur et al., 2006) as well of RLP068/Cltodevelopresistantmutantswithoutillumination
asinpatientswithlong-termoralcarriageharborhigh-persister was evaluated. It was demonstrated that 20 consecutive APDT
mutants(Lafleuretal.,2010).Thisconceptmayexplainpartially treatmentswithRLP068/Cldidnotresultinanyresistantmutants
themechanismofactioninthesuccessofmiconazoletoaugment andthat,indarkconditions,onlyS.aureus strainshadincreased
PDI-mediatedbytheporphyrinTMP-1363andMBinC.albicans minimallyinhibitoryconcentrations(MICs)ofRLP068/Cl.How-
(Snelletal.,2011).Inthisstudy,alistofantifungalsweretested, ever,eveninthiscase,thesusceptibilityof themutatedbacteria
miconazoleandketoconazolebothstimulatedROSproductionin toAPDTwasnotaffectedbytheirMICincrease.Ehrenshaftetal.
C.albicans,butonlymiconazoleenhancedthekillingofC.albicans (1998)showedthattheSOR1(singletoxygenresistance1)gene
andinducedprolongedfungistasisinorganismsthatsurvivedPDI. present in Cercospora fungi plays a role in the resistance to PS
Althoughthedatasuggestedthatpotentiationof antifungalPDI generating singlet oxygen. Studies with SOR1 deficient mutants
bymiconazoleislikelytobemulti-factorial(Snelletal.,2011)and resulted in cercosporin and PS sensitivity. However, the func-
themodelstestedwerenotrelatedwiththebiofilmphenotypes. tion of the protein encoded by this gene and the mechanisms
involvedinCercosporatoxinauto-resistanceremainunclear.PDI
FROMTHEANTIFUNGALPDIMECHANISMTOTHE withRBintheyeastS.cerevisiae,demonstratedaroleofYap1pand
QUESTIONOFRESISTANCEDEVELOPMENT Skn7p in the defense against singlet oxygen (Brombacher et al.,
Themechanismof photodynamicactionhavebeendescribedas 2006).
multi-factorial and non-specific (Bertoloni et al., 1989; Gonza- Superoxidedismutaseisupregulatedfollowingprotoporphyrin-
lesandMaisch,2012).Itinvolvesthedamageof fungalcellwall mediated PDI in S. aureus and RB-mediated PDI in S. mutans
and membrane by ROS. Subsequently, the intra cellular oxidiz- inducesthebacterialheatshockproteinGroEL–responsiblefor
ing species generated by light excitation induce photodamage refoldingdenaturedproteinstonativeconformationsandstabi-
to multiple cellular targets. The event list includes inactivation lizinglipidmembranesduringstress(Nakoniecznaetal.,2010).
of enzymes and other proteins, peroxidation of lipids, leading TheseobservationsareinaccordancewiththoseofSt.Denisetal.
to the lyses of cell membranes, lysosomes, and mitochondria (2011b)whodemonstratedthatsub-lethalPDIstressincreasedthe
and finally causing cell death (Gonzales and Maisch, 2012). A expressionofthetwomajorbacterialheatshockproteinsGroEL
recent study employing the PS Pc 4 in planktonic C. albicans andDnakandthatexposingE.coliandE.faecalistoheatpretreat-
cellshighlightedtheaforementionedproposedsequenceofevents. mentpriortoPDI(apositiveregulatorofGroEL)conferredstress
Furthermore, changes in nuclear morphology characteristic of tolerance,increasingE.coli cellviabilityby2logsandE.faecalis
apoptosis,whichweresubstantiatedbyincreasedexternalization cellviabilityby4logs.
ofphosphatidylserineandDNAfragmentationfollowingPc4-PDI
suggestingthatapoptoticphenomenawererelatedwithfungalcell ANTIMICROBIALPDT:FROMBENCHTOPTOBEDSIDE
death(Lametal.,2011). Withtheresultsfrominvitro studiesbeingpromisinginawide
Since disruption of electron transport chain (ETC) function array of fungal species, a number of clinical applications for
increasesintracellularlevelsofROSinyeast,interferencewithETC antimicrobialPDThavebeentestedandperformedinvivo.PDT
assembly, or function will enhance antifungal PDT. The meta- hasbeenproposedformanydentalapplicationsduetotheacces-
bolic inhibitor antimycin A and defined genetic mutants were sibility of the oral cavity. Nevertheless, the complexity of oral
usedtoidentifyETCcomponentsthatcontributetothesensitiv- microflora makes this microenvironment quite challenging for
itytoTMP-1363-PDTinC.albicans,C.glabrata,andS.cerevisiae thedeploymentofnovelantimicrobials.Aninvivostudyusingan
(Chabrier-Rosellóetal.,2010). immunodeficientmurinemodelwithoralazole-resistantcandidi-
The studies and reports discussing the potential of microbes asis and topical treatment employing 500mg/ml MB combined
to develop resistance to PDT are scattered, quite controversial, with red light totally inactivated C. albicans in the oral cavity
and with a few exceptions they involve bacterial species. The and even prevented the emergence of resistance (Teichert et al.,
non-selective nature of APDI appears as a competitive advan- 2002).AsecondstudyemployedimmunosuppressedSwissmice
tage in the activation of a specific microbial resistance path- orallyswabbedwithC.albicans.Fourdaysafteroralinoculation,
way. In a conventional biological study of routine stress fol- PDT was performed on the dorsum of the tongue after topical
lowed by re growth, 5,10,15-tris(1-methylpyridinium-4-yl)-20- administrationofPhotogem®andfollowedbyilluminationwith
(pentafluorophenyl)-porphyrintriiodide(Tri-Py(+)-Me-PF)was LEDlightat455or630nm.DeterminationofCFU,histological,
employedasPSagainstVibriofischeri andE.coli.Aftertencycles andinflammatoryresponseevaluationofthesurgicallyremoved
of partialinactivationfollowedbyregrowth,neitherof thebac- tonguesaftereuthanasia(Mimaetal.,2010)revealedeffectiveness
teriadevelopedresistancetothephotodynamicprocess(Tavares oftheapproach.
et al., 2010). In a similar study, Giuliani et al. (2010) investi- The very nature of PDT makes it ideal for the treatment of
gated the potential of phthalocyanine RLP068/Cl mediated PDI skin, wound, and burn infections, all of which are easily acces-
to induce resistance to the Gram-positive S. aureus, the Gram- sible for topical application of PS and light. Dai et al. (2011b)
negative P. aeruginosa,and C. albicans,using both sensitive and reportedPDTusingphenothiaziniumsforprophylaxisandtreat-
resistantstrains.Itsability,followingactivationbylight,toinduce mentofcutaneousC.albicansinfectionsinmice.Amousemodel
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Daietal. Antifungalphotodynamictherapy
ofskinabrasioninfectedwithC.albicanswasdevelopedbyinoc- Photodynamic therapy also has promising potential in the
ulatingwoundswithaluciferase-expressingstrainandreal-time treatment of superficial fungal skin infections caused by der-
monitoringoftheextentofinfectionnon-invasivelythroughbio- matophytes. T. rubrum is responsible for tinea pedis (athelete’s
luminescence imaging. In vitro PDI studies showed that (NMB; foot), fungal folliculitis, onychomycosis, and dermatophytosis
Figure2B)wassuperiortoTBOandMB.PDTinvivo initiated (tinea or ringworm). Employing an ex vivo infection model of
either at 30min or at 24h post infection significantly reduced humanstratumcorneumofT.rubrum,Smijsetal.(2009)incu-
C. albicans burden in the infected mouse skin abrasion wounds batedsampleswiththePS5,10,15-tris(4-methylpyridinium)-20-
(Figure6;Daietal.,2011a). phenyl-(21H,23H)-porphinetrichloride(SylsensB)anddeutero-
C.albicansearpinnainfectionusingamousemodelwastreated porphyrinmonomethylester.Uponlightapplication,bothPSwere
with0.3mg/mlporphyrinTMP-1363appliedtopicallyincombi- showntobeactiveantifungals.Moreover,5-aminolevulinicacid
nationwith90J/cm2 greenlight.Additionally,thephototoxicity (5-ALA) and red light has an effect in the treatment of ony-
of both TMP-1363 and MB against C. albicans was compared chomycosis (Donnelly et al., 2005; Kumar and Kimball, 2009).
invitro.TMP-1363uponirradiationatafixedfluenceof2.4J/cm2 Treatmentofrefractoryfingernailonychomycosiscausedbynon-
causedmorethanthreelogsofC.albicans inactivationthanMB dermatophytemoldswithmethylaminolevulinatePDT(Gilaberte
at10mMincubationconcentrationforbothPS.However,TMP- etal.,2011).
1363 was inefficiently photo excited, since its peak absorption Chromoblastomycosis is an infection that involves skin and
did not coincide with the irradiation lamp source, underesti- subcutaneous tissues caused by the traumatic inoculation of
mating the PDI efficacy of TMP-1363. On the other hand, MB dematiaceous fungi species, being that the most prevalent are
absorption peak was compatible with the emission of the lamp Fonsecaea pedrosoi and Cladophialophora carrionii. A clinical
source.ConsequentlythenumberofphotonsabsorbedbyTMP- PDT study employing MB as the PS and a LED device as
1363wasapproximately10-foldlowerthanthatabsorbedbythe light source for the treatment of chromoblastomycosis showed
equivalent concentration of MB (Mitra et al.,2011) It was con- promising results (Lyon et al., 2011). PDT for distal and lat-
cludedthatTMP-1363displayedamoreeffectivemicrobialkilling eral subungual toenail onychomycosis caused by T. rubrum
thanMBduetodifferencesincellularuptakeand/orintracellular have been evaluated in single-center open trial (Sotiriou et al.,
localization. 2010).
FIGURE6|(A)Successivebioluminescenceimagesofarepresentative abrasioninfectedwith107CFUC.albicans,showingthepresenceofyeasts
mouseskinabrasioninfectedwith107CFUC.albicans.(B,C)Representative [(B),arrows],hyphalfilaments[(C),circle],andinflammatoryinfiltrate(C).
periodicacid-Schiff-stainedskinbiopsyspecimentakenfromamouseskin Biopsywastakenonday1postinfection.Bar,20μm.
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