Potential applications of porphyrins in photodynamic inactivation beyond the medical scope

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ContentslistsavailableatScienceDirect

Journal

of

Photochemistry

and

Photobiology

C:

Photochemistry

Reviews

j ourn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / j p h o t o c h e m r e v

Review

Potential

applications

of

porphyrins

in

photodynamic

inactivation

beyond

the

medical

scope

Eliana

Alves

a

_,

_Maria

_A.F.

_Faustino

b

_,

_Maria

_G.P.M.S.

_Neves

b

_,

_Ângela

_Cunha

a

_,

Helena

Nadais

c

_,

_Adelaide

_Almeida

a,∗

a_Department_of_Biology_and_CESAM,_University_of_Aveiro,_Campus_de_Santiago,_3810-193_Aveiro,_Portugal b_Department_of_Chemistry_and_QOPNA,_University_of_Aveiro,_Campus_de_Santiago,_3810-193_Aveiro,_Portugal

c_Department_of_Environment_and_Planning_and_CESAM,_University_of_Aveiro,_Campus_de_Santiago,_3810-193_Aveiro,_Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4February2014

Receivedinrevisedform29August2014 Accepted3September2014

Availableonline21September2014 Keywords: Porphyrin Photosensitizer Photodynamicinactivation Disinfection Nanomaterials Self-cleaningmaterials

a

b

s

t

r

a

c

t

Althoughthediscoveryoflight-activatedantimicrobialagentshadbeenreportedinthe1900s,onlymore recentlyresearchworkhasbeendevelopedtowardtheuseofphotodynamicprocessasanalternative tomoreconventionalmethodsofinactivationofmicro(organisms).Thephotoprocesscausescelldeath throughirreversibleoxidativedamagebyreactiveoxygenspeciesproducedbytheinteractionbetween aphotosensitizingcompoundandalightsource.

Withgreatemphasisontheenvironmentalarea,photodynamicinactivation(PDI)hasbeentestedin insecteradicationandinwaterdisinfection.Lately,otherstudieshavebeencarriedoutconcerningits possibleuseinaquaculturewatersortothecontroloffood-bornepathogens.Otherpotentialapplications ofPDIinhousehold,industrialandhospitalsettingshavebeenconsidered.

Inthelastdecade,scientiﬁcresearchinthisareahasgainedimportancenotonlyduetogreat develop-mentsintheﬁeldofmaterialschemistrybutalsobecauseoftheseriousproblemoftheincreasingnumber ofbacterialspeciesresistanttocommonantibiotics.Infact,thedesignofantimicrobialsurfacesor self-cleaningmaterialsisaveryappealingideafromtheeconomic,socialandpublichealthstandpoints.Thus, PDIofmicro(organisms)representsapromisingalternative.

Inthisreview,theeffortsmadeinthelastdecadeintheinvestigationofPDIof(micro)organisms withpotentialapplicationsbeyondthemedicalﬁeldwillbediscussed,focusingonporphyrins,freeor immobilizedonsolidsupports,asphotosensitizingagents.

Contents

1. Introduction... 35

2. Applicationsontheenvironment,waterandfoodstuff... 36

2.1. Insectpestelimination... 36

2.2. Waterdisinfection... 44

2.3. Eliminationoffood-bornepathogens... 48

3. Applicationsfordomestic,industrialandhealthcaresettings... 50

3.1. Porphyrin-embeddedfabric... 50

3.2. Porphyrin-embeddedpaper... 51

3.3. Porphyrinsimmobilizedinothersupportmaterials... 51

4. Conclusions... 53

Acknowledgements... 53

References... 53

∗ Correspondingauthor.Tel.:+351234370784. E-mailaddress:aalmeida@ua.pt(A.Almeida).

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ElianaAlveswasborninPorto(Portugal).Shereceived herM.Sc.degreein2007andherPh.D.degreein Biol-ogyin2013,bothfromtheDepartmentofBiologyand CESAMoftheUniversityofAveiro(Portugal).Duringseven years,herresearchworkwastotallydedicatedto Photobi-ology,namelytomicrobialphotodynamictherapy,using cationicporphyrinsasphotosensitizers.Atthemoment, sheisaPostdoctoralResearchFellowattheDepartment ofChemistryandQOPNAoftheUniversityofAveirowhere sheisacquiringexpertiseinlipidomics,anexpandingﬁeld oflipidanalysisthroughchromatographicandmass spec-trometrytechniques.

Maria Amparo F. Faustino received her doctoral degree in Chemistry from University of Aveiro, in 1999. She is currently working as Assistant Profes-sor in the Department of Chemistry, University of Aveiro. Hercurrentareaofresearch includes synthe-sisandtransformationoftetrapyrrolicderivativesand their applications, mainly environmentalapplications. http://orcid.org/0000-0003-4423-3802

MariadaGrac¸aP.M.S.NevesisAssociatedProfessorwith HabilitationattheDepartmentofChemistryofthe Univer-sityofAveiro(UA).SheobtainedherHabilitationandPh.D. degreefromUA,herM.Sc.degreefromUMIST, Manch-ester(GreatBritain)andherB.Sc.degreeinChemistry byUniversityofLourenc¸oMarques(Mozambique).Her researchinterestsarecenteredonthesynthesis, func-tionalizationandpotentialapplicationsoftetrapyrrolic macrocycles likeporphyrins,corrolesand phthalocya-nines.https://orcid.org/0000-0002-7953-8166

ÂngelaCunhaisAssistantProfessorattheDepartmentof BiologyandresearcheroftheCenterforEnvironmental andMarineStudies(CESAM)oftheUniversityofAveiro (Portugal).ShereceivedherPh.D.degreeinBiologyin 2001,attheUniversityofAveiro,andhasbeenworking onenvironmentalmicrobiologyandmicrobialecologyof aquaticenvironments.Herrecentinterestsarethestudy ofthe effectofbiosurfactants onthe developmentof microbialbioﬁlmsandonofphotodynamicapproachesfor thepreventionandcontrolofmicroorganisms,witha par-ticularfocusonresistantstructures,suchasendospores, andpolimicrobialbioﬁlms.

M.HelenaG.A.G.NadaisisAssistantProfessoratthe DepartmentofEnvironmentandPlanninginthe Univer-sityofAveiro(UA).SheisaChemicalEngineerholdinga MSc.inChemicalEngineering(1993)fromInstituto Supe-riorTecnico,TechnicalUniversityofLisbon,Portugal,and aPh.D.(2002)inEnvironmentalSciencefromtheUA.Her researchinterestsarecenteredonwatertreatmentand onmaterialandenergeticvalorizationofwastewatersand wastes.Shehasmorethan60publications.Shehas partic-ipatedwhetherasteammemberorasmaininvestigatorin variousscientiﬁcresearchprojectsandrecently submit-tedapatentapplication.Sheisfullmemberoftheresearch centerCESAM(www.cesam.pt).

AdelaideAlmeidaisAssistantProfessoratthe Depart-mentofBiologyfromtheUniversityofAveiro(Portugal), whereshegotherPh.D.degree,in2001.Sheisan inte-gratedmemberoftheAssociatedLaboratoryCentrefor EnvironmentalandMarineSciences(CESAM).Inthelast years,shehasbeeninvolvedinthedevelopmentand applicationofalternativemethodstotheuseof antibi-otics,suchasphotodynamictherapyandphagetherapy. Shehaspublishedintheseﬁeldsandherpublicationscan befoundinhttp://www.cesam.ua.pt/adelaidealmeida.

1. Introduction

Photodynamictherapyreferstotheuseofalightsource(visible

lightofanappropriatewavelength),anoxidizingagent(molecular

oxygen,O2)andanintermediaryagent(namedphotosensitizer,PS)

abletoabsorbandtransfertheenergyofthelightsourceto

molec-ularoxygenleadingtotheformationofhighlycytotoxicspecies

(singletoxygen[1_O

2],hydrogenperoxide[H2O2],and/orfree

radi-cals,suchassuperoxide[O2−•]andhydroxylradical[HO•]),causing

a multi-targeteddamage and destructionofliving tissues[1,2].

Thegenerationofthesereactiveoxygenspecies(ROS)canoccur

viatwo mechanismsorpathways, knownastypeI andtype II,

whichrequirethepresenceofO2(Fig.1).Inthepresenceoflight

(h),thephotosensitizerinthesingletgroundstateabsorbsa

pho-ton,affordingtheexcitedsingletstate.Then,itcanloseenergyby

returningtothesingletgroundstatewithﬂuorescenceemission

(F)or,throughanintersystemcrossing(ISC)process,itcanbe

con-vertedinthelong-livedtripletstate.Thisexcitedtriplet-statePS

candecaytogroundstatebyphosphorescenceemission(P)orcan

reactwithasubstrate,namelyanelectrondonormolecule.Inthis

casetheformationofradicalionscanoccurgivingrisetoradical

ionswhichreactwithgroundstateoxygen(3_O

2),originatingROS

(typeImechanism).Alternatively,theexcitedtriplet-statePScan

transferenergydirectlytomolecularoxygenaffordingtheexcited

singletstate(1_O

2)(typeIImechanism).Bothphotoprocessesmay

occursimultaneouslybuttypeIIis,ingeneral,thepredominantone.

Thecytotoxicspeciescancauseirreversibledamagetoproteins,

nucleicacidsandlipids[3,4].

Theadvantageofbeingaprocesswithoutaspeciﬁccelltarget

rendersphotodynamicinactivation(PDI)effectiveintheoxidation

ofdifferentbiomoleculeswiththeconsequentdestructionof

sev-eralcelltypes.Infact,thismethodologyhasabroadspectrumof

activityand, usingthe samePS, is abletodestroy humancells

[1], viruses[5],bacteria [6],molds [7],yeasts[8],protozoa[9],

helminths[10]andinsects[11].

Moreover,theabilitytostructurallytailorthePSaswellasto

successfullylinkittoothermolecules,withahighdegreeof

speci-ﬁcity(e.g.,antibodies,enzymes,nucleicacids),ortosolidsupports

givesthistherapyamultiplicityofclinicalandnon-clinical

appli-cations.

The discovery that positively charged PS could effectively

inactivateGram-negativebacteriawithouttheadditionof

perme-abilizingagents[12,13]broughtanewimpetustotheinvestigation

on the PDI of microorganisms as a new therapeutic

modal-ity.

Thedifferenceinsusceptibilitybetweenthetwotypesof

bac-teria,Gram-negativeandGram-positive,isexplainedonthebasis

ofthestructuralfeaturesoftheircellwall(Fig.2).Gram-positive

bacteria havea cell wallcomposedof lipoteichoicand teichoic

acidsorganizedinmultiplelayersofpeptidoglycan,whichconfers

adegreeofporositytobacteriasoastofacilitatetheanchoring

and entry of PS into the cell [14,15]. In Gram-negative

bacte-ria,thepresenceofacomplexoutermembraneinthecellwall,

consistingofphospholipids,lipopolysaccharides,lipoteichoicacids

andlipoproteinscreatesanimpermeablebarriertoantimicrobial

agents [14,15].TheinteractionbetweenthecationicPSand the

constituentsoftheGram-negativecellwallgenerateselectrostatic

interactionsthatpromotedestabilizationofthenative

organiza-tionofthewall,allowingthebindingandeventualentryofthePS

moleculesintothecell[14,15].Inthecaseoffungi,thecellwall

containschitin,glucansandlipoproteinsthatrepresentabarrier

withintermediatepermeability incomparisontoGram-positive

andGram-negativebacteria[16].Withregardtoviruses,enveloped

virusesaremoreeasilyinactivatedthannon-envelopedones,but

somestudiesshowthatnon-envelopedvirusescanalsobe

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Singlet ground statePS Singlet excited state PS Triplet excited state PS Dioxygen Substrate Reacve oxygenspecies Singletoxygen

Irreversibledamageon

major cellcomponents

TypeI mechanism TypeII mechanism h ν F ISC P IC ISC

Fig.1.Schematicrepresentationofthephotoprocessesthatcanoccurduringphotodynamicinactivation.ISC,intersystemcrossing;IC,internalconversion.

beingtheefﬁciencyoftheirinactivationsimilartothatof

Gram-negativebacteria[18].

In recent years, the synthesis of new compounds for PDI

hasgrowndramatically,manyofthemwithverygood

inactiva-tionresults.SeveralclassesofPS,suchasphenothiaziniumdyes

(methyleneblue,toluidineblueO),naturallyoccurringPS

(chloro-phylls, psoralens, perylenequinonoid pigments), tetrapyrroles

(porphyrins, phthalocyanines, chlorins, bacteriochlorins) and

fullereneshavebeensuccessfullytested[19–21](Fig.3).

Thegroupofporphyrinderivatives hasbeenprominent, not

onlybecauseitincludestheﬁrstformulationapprovedfor

pho-todynamictherapyofcancer[22]but alsointheperspectiveof

environmentalapplications,consideringthattheuseofnaturally

occurringporphyrinsorsyntheticrelatedanalogsarisesasan

eco-nomical,eco-friendlyandhuman/animalsafeoption[23].

Porphyrinsareaclassofheterocyclicaromaticcompounds

con-stitutedbyfoursubunitsofpyrroletypelinkedbymethynicbridges.

Thepresenceofthesecompoundsisubiquitousinnatureaspartof

vitalbiochemicalprocessessuchasoxygentransport(hemegroup)

andphotosynthesis(chlorophylls)[24].

Ifphotodynamictherapyisnowanestablishedprocedurefor

thetreatmentofcertainnon-oncologicalandoncologicaldiseases

[22,25,26],itisstillnotusedforinfectiontreatment[15,27].In

addi-tion,thepotentialapplicationofPDItodestroy(micro)organisms

goesbeyondthemedicalﬁeld,withparticularfocusonthe

envi-ronmentalarea[28–32].

InPDIstudiesrelatedwithnon-clinicalapplications,artiﬁcial

white light (halogen or xenon lamps) and sunlight have been

usedinordertoachieveorganisms’destruction.Theirradiance(or

ﬂuencerate)canbegiveninWm−2,inlux(lx)orin␮Em−2s−1

(withEstandingforEinsteinsintheformerterminology, which

hasbeenreplacedinthenewterminologybyM,meaning“mole

oflight”).Theselightunitscanbeinterconvertedinthefollowing

way: 1lx≈9.5×10−3mWcm−2≈1.8×10−3␮Mm−2s−1 [33].

Forexample,100–150lxmayrepresentashadyroominnatural

light,30,000–40,000lxanovercastsummer’sdayand100,000lxa

verybrightsummer’sday[34].Sincetheexperimentalconditions

describedinliteraturereportsarequitedifferent,sometimesthe

resultscannotbedirectlycompared.Thesamelightdose(Jcm−2)

canbeachieved byvarying thelight irradiance, theirradiation

timeorboth[21].However,theeffectivenessoftheresultsmaybe

differentwhenusingahighirradianceoverashorttimeperiodora

lowirradianceoveralongertime,eventhoughthelightdoseisthe

sameineachcase[21,35].Ingeneral,thePDIofmicroorganisms

ismoreextensivewhenhigher irradianceandlongertreatment

durationareused[36].Increasingthedurationofirradiationwill

improvetheyieldoftreatment,compensatingalowconcentration

ofPSoralessefﬁcientPStype[37].

Duetoitsmulti-targetnature,andtherefore lowprobability

oftriggeringthedevelopmentofresistancein(micro)organisms

[38–41],this therapy hasbeentested invarious researchareas

as an alternativeapproach toactual methods tocontrol insect

pests,waterquality,microbiologicalfoodquality;andalsointhe

disinfectionand sterilizationofmaterialsandsurfacesin

differ-entcontexts(industrial,householdandhospital).Furthermore,the

useof this formof eradicating microbes or noxious organisms

becomesincreasinglyachievableinpracticeifonethinksthat,for

certainpurposes,thePSmaybeimmobilizedoninertsolid

sup-portsallowingtheirreuseandrecycling,makingthistechnologyof

broad-spectrumactivity,economic,sustainable,durable,and

envi-ronmentallyfriendly.Tothisextent,withthegreatdevelopment

ofnanotechnologyandmaterialschemistry,severaldifferent

sup-portshavebeencreatedtoimmobilizeaseriesofPSdesignedfor

photoinducedoxygenationreactions[42].

Theaimofthisreviewistopresenttheeffortsmadeinthelast

decadeintheinvestigationofPDIof(micro)organismsbeyondthe

medicalﬁeld,usingporphyrinsasPS,eitherfreeorimmobilized

insolidsupports.AlltheorganismswhichhavebeenusedonPDI

experimentsarelistedinTable1.TheporphyrinicPSreportedin

thesestudiesarelistedinTable2.

2. Applicationsontheenvironment,waterandfoodstuff

2.1. Insectpestelimination

Alternativepesticides for pestand vector control havebeen

requiredformorethanadecade.Insecticideindustryandvector

control management face several problems nowadays:

devel-opmentof resistanceinmajor vectorstocommoninsecticides;

abandonmentofcertaincompoundsforsafetyreasonsand

envi-ronmentalandhumanhealthconcernsabouttheuseofmanyolder

generationinsecticides,suchasDDT;economicfactorsandmarket

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Table1

Listoforganismsusedinnon-clinicalphotodynamicinactivationexperiments.

Organism Organismtype References

Acinetobacterbaumannii Bacterium(Gn) [43]

Acremoniumspp. Fungus(mold) [44]

Acremoniumstrictum Fungus(mold) [45]

Aedesaegypti Mosquito(denguevector) [46,47]

Aedescaspius Mosquito [48]

Aeromonassalmonicida Bacterium(Gn) [49]

Alternariaalternata Fungus(mold) [45]

Alternariaspp. Fungus(mold) [44]

Anophelesarabiensis Mosquito(malariavector) [50]

Anophelesgambiae Mosquito(malariavector) [50]

Anophelessp. Mosquito(malariavector) [51]

Artemiafranciscana Crustacea(Branchiopoda) [52]

Ascarislumbricoides Helminth [36]

Aspergillusﬂavus Fungus(mold) [7]

Aspergillusspp. Fungus(mold) [44]

Bacilluscereus Bacterium(Gp) [53–58]

Bactroceraoleae Fly(oliveﬂy) [59]

Baculovirus EnvelopedDNAvirus [60]

Candidaalbicans Fungus(yeast) [61–63]

Ceratitiscapitata Fly(Mediterraneanﬂuitﬂy) [59,64]

Chaoboruscrystallinus Insectlarvae(Diptera) [23,65]

Chaoborussp. Insectlarvae(Diptera) [66]

Colpodainﬂata Protozoan(Ciliophora) [52]

Culexpipiens Mosquito [67,68]

Culexquinquefasciatus Mosquito [46]

Culexsp. Mosquito [66]

Cultivablebacteriafromaquaculturewater Bacteria [49]

Daphniasp.,Daphniamagna Crustacea(Branchiopoda) [52,66]

Edwardsiellaictaluri Bacterium(Gn) [69]

Enterococcusfaecalis Bacterium(Gp) [18,49,70]

Escherichiacoli Bacterium(Gn) [18,41,49,63,70–92]

Fecalcoliforms Bacterium(Gn) [37,93]

Fecalenterococci Bacterium(Gp) [37,93]

Flavobacteriumcolumnare Bacterium(Gn) [69]

Fusariumavenaceum Fungus(mold) [7,45]

Fusariumculmorum Fungus(mold) [94]

Fusariumpoae Fungus(mold) [94]

Fusariumspp. Fungus(mold) [44]

Ichthyophthiriusmulftiﬁliis Protozoan [65,95]

InﬂuenzavirusstrainX-31,A/Aichi/2/68(H3N2) Virus [96]

Legionellapneumophila Bacterium(Gn) [97,98]

Liriomyzabryoniae Fly(leafminerﬂy) [99]

Listeriamonocytogenes Bacterium(Gp) [53,57,80,100–103]

Mucorspp. Fungus(mold) [44]

Muscadomestica Fly(houseﬂy) [68]

Myceliasterilia Fungus(mold) [44]

Mycobacteriumsmegmatis Bacterium(Acid-fast) [75]

Parasarcophagaargyrostoma Fly(ﬂeshﬂy) [104]

Penicilliumchrysogenum Fungus(mold) [105]

Penicilliumspp. Fungus(mold) [44]

Photobacteriumdamselaesubsp.damselae Bacterium(Gn) [49]

Photobacteriumdamselaesubsp.piscicida Bacterium(Gn) [49]

Planktothrixperornata Cyanobacterium [69]

Polyomavirus Virus(Non-envelopedDNAvirus) [60]

Pseudomonasaeruginosa Bacterium(Gn) [43,83]

Pseudomonassp. Bacterium(Gn) [49]

Rhizopusoryzae Fungus(mold) [7,45]

Rhizopusspp. Fungus(mold) [44]

Saccharomycescerevisiae Fungus(yeast) [8]

Salmonellaenterica Bacterium(Gn) [102,106]

Salmonellasp. Bacterium(Gn) [80]

Saprolegniaspp. Fungus(mold) [81]

Selenastrumcapricornutum Greenalga [69]

Staphylococcusaureus Bacterium(Gp) [43,49,69,73–75,77–83,86,87,91,107,108]

Stomoxiscalcitrans Fly(houseﬂy) [109]

T4-likephage Bacteriophage [17,38,70]

Taeniasp. Helminth [36]

Tetrahymenathermophila Protozoan(Ciliophora) [52]

Trichotheciumroseum Fungus(mold) [7]

Ulocladiumoudemansii Fungus(mold) [8]

Vibrioanguillarum Bacterium(Gn) [49]

Vibrioﬁscheri Bacterium(Gn) [110,111]

Vibrioparahaemolyticus Bacterium(Gn) [49]

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Fig.3. Classesofnaturalandsyntheticphotosensitizersusedinphotodynamicinactivationofmicroorganisms.

constraints;lackofavailable,safeandcost-effectivelarvicidesfor

additiontodrinkingwater; depletionof safeand cost-effective

insecticides;re-emergenceofdiseasecausedbytheearlyeffortsto

reduceindoorresidualinsecticideapplicationratesandincrease

ofvector-bornediseasetransmission[112–114].

Therefore,thereisanurgentneedforimproved,novel,

cost-effectiveinsecticidesandinsecticidetreatedmaterials;long-lasting

insecticideformulationsforsuchmaterialsandsprays;economic

feasibility and commercialization analysesfor pesticides

devel-opmentand,mostimportantly,providingsolutionstoovercome

the increasing problems of resistance to current insecticides.

Accordingly,it has beensuggested thedevelopmentof

insecti-cidecombinations,suchasbi-treatednetsandinsecticidetreated

materials[112],ornewactiveingredientsbasedonnovelmodesof

action[115].

TheideaofusingPSasphotopesticides(orphotoinsecticides)

isnotnew[116,117].However,theuseofporphyrinsforthis

pur-posewasonlymentionedfromthelate1980softhelastcentury

[118–121].Sincethen,theuseofnaturalorsyntheticporphyrin

derivativeshasbeenincreasinglyexploitedtocontroland

eradi-catevarioustypesofinsects,includingpestﬂiescapableofinducing

signiﬁcantdamagetoagriculturalcrops,andmosquitoesvectorsof

pathogensresponsibleformalaria(Anopheles),yellowfever(Culex,

Aedes)denguefever(Aedes)andencephalitis(Aedes,Culex,

Anophe-les)[46–48,50,66–68].Nowadays,thereisaglobaldistributionof

these organisms. In the case of Aedes aegypti, for example, its

sporadicpresencehasbeenrecognizedin mid-latitudessuchas

in Britain,France, Italy, Spain, Malta,Portugal and other

Euro-peancountries[122,123].Recently,inMadeiraisland(Autonomous

RegionofMadeira),Portugal,therewasadengueoutbreakstarting

inOctober2012with2170casesofdenguefevernotiﬁeduntilearly

April2013.InmainlandPortugal,11caseshavebeenreportedand

71casesin13Europeancountries,allintravelersreturningfrom

theisland,andnoneofthemlethal[124].Currentupdatesofﬁrst

detectionorconﬁrmationofthepresenceofotherdiseasevectors

canbefoundattheEuropeanMosquitoBulletinwebsite[125].

Inthephotosensitizedinsecteradication,PSwhicharealready

registeredasfoodadditives,suchaschlorophylls,chlorophyllins

andtheircoppercomplexes[126,127],orphototherapeuticagents

ashematoporphyrinIX(HpIX)or5-aminolevulinicacid(5-ALA),

used as a precursor of protoporphyrin IX (PPIX) [22], have

been especially successful. They have a low cost production,

lack of mutagenic activity, high efﬁciency and high level of

safety tohumans and, in general,to othermammalian species

[22,128].

Typically,experimentaldesignsoflaboratorystudies,or

stud-iesundersemi-ﬁeldconditions,useﬂylarvaeoradultmosquitoes,

laboratory-rearedorcollectedinbreedingsites,whicharebrought

into contact withsolutions containing free-base porphyrins or

porphyrinincorporatedintobaits.Aninitialperiodofdark

incu-bation,moreorlessextendedtoallowtheuptake/ingestionofPSis

followedbyirradiationwithnaturalorartiﬁcialsunlightand

deter-minationofthemortalitypercentageorthemedianlethaldose

(LD50,i.e.,thedoseatwhichdeathisproducedin50%ofthe

exper-imentalorganisms)(Fig.4).Manystudiesalsoinspecttheamount

ofporphyrintakenup,theanatomicalsitewhereitbindsandits

clearance.

HematoporphyrinIX(HpIX)hasbeentestedonCeratitis

capi-tata(Mediterraneanfruitﬂy)andBactroceraoleae(oliveﬂy)[59].A

sugar/proteinbaitcontaining8␮MHpIXledto100%mortalityofC.

capitataandB.oleaeﬂieswithintheﬁrstdayafter1and2hof

expo-sureto2080␮Es−1m−2,respectively.However,amilderirradiance

suchas760␮Es−1m−2wasalsofoundeffectiveindecreasingthe

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to2h.ThelowerphotosensitivityofB.oleaeﬂieswaspossiblydueto

thesmalleramountofingestedHpIXand/ortoitsdarker

pigmenta-tion.HpIXwaslocalizedinthecuticle,themidgut,theMalpighian

tubesand theadiposetissueof theﬂies[59].ThesamePSwas

alsotestedonStomoxiscalcitrans(houseﬂy)[109].Fliesfedwith

aconcentrationrangefrom4.7to7.5␮MofHpIXandirradiated

for1hatanirradianceof1220␮Es−1m−2underwenttotal

mor-talityafter2–3days.OnlyatthehighestHpIXdose,asigniﬁcant

percentageofdeadﬂieswasobserved,forthe1hoflight

expo-sure.Thisinsecthasamoredarklypigmentedbodyandlargersize

thanC.capitataandB.oleae.BesidesHpIX,othermeso-substituted

porphyrins,withtwo,threeandfourpositiveornegativecharges

havebeenusedforinsectphotosensitization[109,129].Themost

efﬁcientporphyrinwas

5,10-bis(1-methylpyridinium-4-yl)-15,20-diphenylporphyin(Di-Py+_-Me-Di-Ph

adj), adicationicamphiphilic

porphyrinwhich at theconcentration of 0.85mM caused 100%

mortality on C. capitata within 1h of irradiation at an

irra-diance of 1220␮Es−1m−2. The photoinsecticidal efﬁciency of

porphyrinsseemstoincreasewiththeincreasinghydrophobicity

of themolecule, eitherby thereduction in theoverall number

ofpositive ornegativechargesor bythereplacementofthe

1-methylpyridiniummoietywithphenylrings.

Thefactorsthatappeartoaffecttheefﬁciencyofinsects’

pho-tosensitivity by porphyrins have been identiﬁed by Ben Amor

etal.[59,109,129],servingasastartingpointfordesigningnew

strategiesfortreatmentoptimizationandspeciﬁcityincrease.Such

factors are light irradiance,total light dose, PSchemical

struc-ture (higher photosensitivity associated with higher degree of

hydrophobicity of the porphyrin, particularlyobvious with the

amphiphilicones),concentrationofporphyrininthebait,

thick-nessandcoloroftheinsectintegument,andclearancefromthe

organismina 24–48htimeinterval aftertheporphyrinuptake

[130].

HpIXandthreemoreporphyrinderivativeswereusedinthePDI

ofdifferentmosquitolarvaeunderﬁeldconditions.AHpIXLD50of

3.2mgL−1wasestablishedforthefourthinstarofCulex

quinque-fasciatusandthisporphyrinwastheonlyeffectiveoneonA.aegypti

larvae[46].

The strong and fast photo-larvicide activityof HpIXagainst

C. capitata immature larval stages has also been reported

Table2

Listofporphyrinicderivativesusedinnon-clinicalphotodynamicinactivationexperiments.

Porphyrin Immobilized(references)

No[47] No[50,52,69]

Onﬁlterpaper[82] Oncottonfabric[86,108]

(7)

Table2(Continued)

Onpolysilsesquioxaneplasticﬁlms[61] Oncottonfabric[87]

Onazide-modiﬁedcellulosenanocrystals[43,75] Onchloroacetylcelluloseesterchlorides[78] Oncelluloselaurateestersplasticﬁlms[79]

No[71] No[93]

No[49,71,93,110]andTri-Py+_-Me-PF_on_silica coatedFe3O4nanoparticles[70]andalsoon CoFe2O4nanoparticles[111]

No[81] No[105]

No[36,37,71,74,81,105];Tetra-Py+_-Me_was entrappedintomicroporoussilicagels[88]and intothreealkylene-bridgedpolysilsesquioxanes [89]

(8)

Table2(Continued)

Onopticallytransparentindiumtinoxide electrodes[63]

Onchitosanmembranes[72]

Pd(II)TetraTPPCO2HOnapolyurethanematrix[83]

No[105]

Inhydrophilicpolycaprolactoneandpolyurethane (TecophilicH)nanoﬁbers[60];onelectrospun polymericnanoﬁbermaterialspolyurethane LarithaneTM_,_polystyrene,_{polycaprolactone,}_and polyamide6[76];intothreealkylene-bridged polysilsesquioxanes[89];andinasilica-gel supportedantimonyporphyrincomplexSbTPP [97,98]

(9)

Table2(Continued)

No[7,8,45,99,59]

No[94];onacid-functionalizedmulti-walled carbonnanotubes[96,107];oncelluloselaurate estersplasticﬁlms[77];andonnylonﬁbers[73]

No[56–58,101–103]andonchitosan[106] (E-140andE-141)Ongelatinﬁlmsandcoatings [80]

No[92];onsilica-gelandonaMerriﬁeld resin-basedmaterial[90]

[64]. The LD50 of HpIX in the food, when activated by light

(47photons␮mols−1m−2), was 0.173mM, determined in the

periodentailedfromegghatchingtoadultecdysis.The

correspond-ingHpIXLD50duringthedispersalperiodalone was0.536mM.

HpIXelicitedamortalityof90.87%,whichwasmainlyconcentrated

duringprepupalandearlypupalstages.Lociinthebrainandinthe

gutweredamagedbyROS[64].

Awadetal. [67]demonstrated theefﬁciencyofHpIXand of

aformulationbasedonHpIXpowder,sugarandotheradditives,

as larvicidal substances onCulex pipiens Egyptian ﬁeld strains,

exposedto3,9and18hofnaturalsunrise.AHpIXconcentrationof

10and100␮Mdecreasedthelarvalsurvivalby94%and99.3%atthe

endof5days,respectively.Ontheotherhand,concentrationsof1

(10)

Fig.4. SchematicillustrationoftheinvivoPDIexperimentsondisease-transmittinginsectvectors,contaminatedfoodstuffandinfectedﬁshinaquaculture.PS:photosensitizer.

of92.7%and99.2%,respectively,after5days.Itwasproposedthat

asynergisticeffectoccurredduetotheincorporationofsugarand

otheradditivestotheHpIX,whichcouldreﬂectthesuitabilityof

usingthesugarasHpIXcarrierinthecommercialformula[67].

El-Tayebet al.[104]studied theeffectof HpIX ontheﬂesh

ﬂyParasarcophagaargyrostomainadultstage.Aconcentrationof

10mMofHpIXcausedamortalityof83%and96%ofthetreated

ﬂiesafterexposuretonaturalsunlightwithanirradianceof236.5

and1935Wm−2,respectively.Histologicalstudiesshowedthehigh

abilityofHpIXtoaccumulatein theinsectorgansandtocause

highextentdamageinthealimentarycanaltissue.Theincrease

inirradianceoflightandinirradiationtimeenhancedﬂy

mortal-ity.So,althoughthemostefﬁcientHpIXconcentrationtocontrol

P.argyrostomawas10mM,concentrationsof1and0.01mMwere

sufﬁcienttocontrolMuscadomesticaandCulexpipiens,respectively

[68].Morerecently,larvaeofthemosquitoAedescaspiushavebeen

efﬁcientlyphotoinactivatedusing1mMofHpIX-formulation.The

larvalmortalityhasimprovedbyincreasinglightirradianceand

exposuretimes[48].

In order to be applied to endemic areas with scarce

eco-nomicresources,PSmustbeinexpensive.Someresearchershave

used natural and modiﬁed PScentered on chlorophyll

deriva-tives(chlorophyllinandpheophorbide)andsunlight.Chaoborussp.,

Daphniasp.andCulexsp.larvaewerephotosensitizedwith

chloro-phyllin(15mgL−1),incubatedindarknessovernightandwerethen

irradiatedfor3–4hwithartiﬁcialsunlight (PAR:149.66Wm−2,

UV-A32.67Wm−2andUV-B0.77Wm−2).Afterincubation,

chloro-phyllin eliminated the different organisms at remarkably low

concentrations. The LD50 value in Culex sp. larvae was about

6.88mgL−1, in Chaoborus sp.larvae of about 24.18mgL−1, and

inDaphnia0.55mgL−1.It wasfoundthat duringthepuparium,

mosquitolarvaearerelativelyinsensitivetochlorophyllin

treat-mentand, duringmetamorphosis,thechrysalidis encapsulated

andtotallystopsfooduptake.Probablybecauseofthat,the

accu-mulationofchlorophyllininsidetheorganismislimitedandthe

photodynamiceffectisreduced.Aswellasinotherstudies,some

dark toxicity hasalsobeen observed.Othersmall animals, like

Daphniaandﬁsh(Chaoborus)larvaearealsoaffectedby

chloro-phyllin[66].Ontheotherhand,unlikeﬁshlarvae,moremature

ﬁshareunharmedandsurvivechlorophyllintreatmentat

concen-trations,atwhich,forexample,Culexlarvaeareseverelyaffected.

This hasbeenshown in ﬁeld testsin Nigeria in which

chloro-phyllinwasusedonAnopheleslarvaeandsuccessfullydestroyed

themosquitolarvaeinatreatedpond,withoutharminganyofthe

otheraquaticorganisms[51].Moreover,inadditiontothe

inactiva-tionofmosquitolarvae,Wohllebeetal.[65]demonstratedforthe

ﬁrsttimethatthephotodynamictreatmentofC.crystallinuslarvae

with24mgL−1ofchlorophyllinsolutiongivesaLC50with0.26MJ

(PAR+UV-A+UV-B)andinducesnecrosisandapoptosisinthese

organisms.Chlorophyllinwasorallytakenupandaccumulatedin

theintestine(adoseof3.2mgL−1chlorophyllinwith3hirradiation

inducedapoptosisintheintestinalcells)[65].

Besideschlorophyllin,magnesiumchlorophyllin,zinc

chloro-phyllandcopperchlorophyllhavebeentestedonC.crystallinus

larvae [23]. After6hof darkincubation and 3hof light

expo-sure(360Wm−2),theLD50valueofmagnesiumchlorophyllinwas

about 22.25mgL−1 and for zinc chlorophyll17.53mgL−1.

Cop-per chlorophyll (LD50 0.1mgL−1) was shown to be toxic also

withoutlight.Chlorophyllin(LD50 14.88mgL−1)waslyophilized

immediately after extraction, and its photodynamic efﬁciency

remainedconstantovera30-dayperiod,representinganincrease

inphotodynamicefﬁciencyof50%comparedtomagnesium

chloro-phyllinisolatedwithstandardmethods(nolyophilization)and18%

compared tozinc chlorophyllin. Theresultsshowed that about

30Wm−2ofsolarradiation,whichis<10%offullsunlightis,was

(11)

30%ofthemosquitolarvae). Dependingontheattenuationin a

waterbody,photodynamicactioncanalsotakeplacebelowthe

watersurface.Temperaturehasalsobeenshowntoinﬂuencethe

activechlorophyllinuptakebythelarvae[23].

AswellasHpIXandchlorophyllin,thehematoporphyrin

deriva-tivedimethyl ether (HpDE) has also beenshown tobe a high

potentialphotopesticideagainstthelarvaeoftheleafminerﬂy

Liri-omyzabryoniae[99].Theinsectsexposedtoasugarbaitcontaining

25mMofHpDEandirradiatedfor30minwithbroadspectrum

visi-blelightatanirradianceof30mWcm−2(54Jcm−2lightdose),died

(100%mortality)1dayafterirradiation(inthecaseofthefemales)

and4 days aftertheirradiation(in thecase ofthemales). The

observeddifferencesinthemortalitykineticsbetweenfemaleand

maleﬂieswerepossiblyduetothebodysizeandbiological

activ-ityoffemales[99].Thetreatmentefﬁciencystrongalsostrongly

dependedonthetypeofPSused,explainingthedifferent

attrac-tivenessoftheinsectsforthebaitaccordingtothespeciﬁcityofthe

PScontainedonit[99].Inlinewiththisneed,differentbaits

con-tainingporphyrintoinactivatedisease-transmittingvectorshave

recentlybeendesignedandtested.

Lucantoni et al. [47] prepared a formulation constituted

by

5-(1-tetradecylpyridinium-4-yl)-10,15,20-tris(1-methylpyridi-nium-4-yl)porphyrin (Tri-Py+_-C

14-Py+-Me) and powdered food

pellet (PFP). First, the LC50 of C14 porphyrin in solution was

tested on photosensitized 3rd–early 4th instar A. aegypti

lar-vae.LC50valuesof0.1␮M(0.15mgL−1)and0.5␮M(0.77mgL−1)

were obtained after irradiation intervals of 12 and 1h, with

4.0mWcm−2 artiﬁcialwhitelight,respectively.Tri-Py+_-C

14-Py+

-Me was shown to be active after ingestion by the larvae and

caused irreversible lethal damage to their intestinal tissues

(midgut and caecal epithelia). The porphyrin carrier

formula-tion (25mg of PFP in 500mL of 50␮M of Tri-Py+_-C

14-Py+-Me

– PF-50-Tri-Py+_-C

14-Py+-Me) and a 5␮M of Tri-Py+-C14-Py+

-Me solution were both 100% effective up to two weeks, and

the amount of Tri-Py+_-C

14-Py+-Me required to prepare the

PF-50-Tri-Py+_-C

14-Py+-Mewas10timessmallerthantheTri-Py+

-C14-Py+-Merequiredtotreattheincubationmedium.Inthesame

direction, Fabris et al. [50] associated

5-(1-dodecylpyridinium-4-yl)-10,15,20-tris(1-methylpyridinium-4-yl)porphyrin (Tri-Py+

-C12-Py+-Me)withtwodistinctcarriers:amodelofapharmaceutical

oralvehicle(Eudragit®_S100,_EU)_and_a_model_of_foodstuff_carrier

(catfoodpelletFriskies®_,_CF)._These_{phorphyrin-carrier}_formulates

(50␮MTri-Py+_-C

12-Py+-Me dose)weretestedagainstovernight

fedAnophelesgambiaeandAnophelesarabiensislarvae,exposedto

sunlight(30–110mWcm−2)for 0.5–3h. Theseconditionsledto

highphotoinactivationefﬁciencyagainstlaboratoryrearedA.

gam-biaeandA.arabiensislarvae(almostcompletemortality).Onwild

(ﬁeld-collected)larvae,theformulationEU-50-Tri-Py+_-C

12-Py+-Me

EUcaused100%mortalityonA.gambiaeMandSformsbut

CF-50-Tri-Py+_-C

12-Py+-Meshowedvariableresultsdependingonthesite

wherethelarvaewerecollected.NotonlytheassociationofthePS

withsuitablecarrierspromotedafastandselectiveinternalization

offormulatesbytheAnopheleslarvaewhichguaranteedtheirdeath

uponsubsequentsunlightexposure,butalsothenatureofthe

car-rieraffectedtheoverallefﬁcacyoftheporphyrinformulates.This

wasshownbytheadministrationofa1:1mixtureofCF-50-Tri-Py+

-C12-Py+-Meandlaboratoryfoodforlarvae(TetraMin®)inducingan

extensivemortalityofbothlaboratoryrearedandwildAnopheles

larvae.Theseresultsindicatedtheprimaryimportanceof

palata-bilityinthedesignoforallarvicideformulations[50].

AccordingtoLucantonietal.[47],aninsolublebaited

insec-ticidalformulateshouldbeactivelyconsumedbythelarvae;an

efﬁcient and cost-effective employment of the PS; suitable for

applicationin householdwater storagesfor drinkingand other

domestic purposes; unlikely to affect the organoleptic

proper-ties of the stored water; selected or manipulated on different

porphyrincarriersinawaytostandardizetheparticledimension

toasizerangethatisespeciallypalatableformosquitolarvae(e.g.,

5–50␮m),thusreducingtherisksofuptakebynon-target

organ-isms.

2.2. Waterdisinfection

Water reuse is increasingly becoming an essential

require-ment.Notonlyin undevelopedcountriesbut alsoin developed

countries,treatingdrinkingwater andwastewatermaybe

chal-lenging.Chlorine-basedagents, themostcommonly usedwater

disinfectants,areeffectiveagainstabroadrangeofmicrobes,are

inexpensiveandeasytouse,butlackeffectivenessagainst

para-sitesandleadtotoxicby-products.Alternativestochlorinesuchas

ozone,chloramines,chlorinedioxide,andUVirradiation,alsohave

advantagesanddrawbacksdealingwithcost,efﬁciency,stability,

easeofapplication,developmentofmicrobialresistanceand

muta-tionstriggeredbyUVirradiation,andnatureofformedby-products

[131,132].Inthisway,newtechnologiesforwatertreatmenthave

emergedinrecentyears[133].

ThePDIofmicroorganismsinthecontextofwatertreatment

isconsideredtocauseminimalenvironmentalimpactand

over-comeeconomic,ecologicalandpublichealthissues.Contrarilyto

theconventionalmethods,notoxicby-productsareformedand

nomicrobialresistanceisdeveloped.Thepossibilityof

immobiliz-ingefﬁcientlyPSininsolublesupportsisalsoanadvantageofPDI.

BythiswayitispossibletoremovethePSaftertreatmentfrom

environmentalwatersandtoreuseit,whichturnsthis

technol-ogyenvironmentallyfriendlyandcost-effective.Besides,theuseof

sunlightaslightsourceinPDIhastobeconsideredinorderto

estab-lishasustainableantimicrobialprotocol.Sincesunlightpenetrates

deeplyintothewatercolumn,anearlyuniformilluminationcan

beachievedandhighwatervolumesmaybetreated.Thisapproach

becomesinexpensivesinceitisbasedontheuseofalowcostvisible

lightsource.Moreover,forPSasporphyrinswiththeSoret

absorp-tionbandinthe420–430nmspectralregion,thereisanefﬁcient

interactionwithbluelight,whichhasahighpenetrationdepthinto

naturalwatersandisthemosteffectiveinthevisiblerangeinthe

inactivationofmicroorganismsbycationicporphyrins[134].

The possibility of treating wastewater for reuse in

agri-culture (crop irrigation) was mentioned in the research work

carried out by Alouini and Jemli [36,37]. Accordingly, the

authors demonstrated the efﬁcient PDI of helminth eggs

by the tetracationic

5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (Tetra-Py+_-Me)_under _visible _light _{illumination,} _in

clearwaterand insecondarytreatedwastewater.Inadditionto

differentconcentrationsofPSandlightintensities,theinﬂuenceof

turbidity(contentofsuspendedsolids),agitation(aeration),

con-centration of dissolved oxygenand theultrastructural changes

oftwotypesofeggsofparasiteswasalsoinvestigated.

Suspen-sionsofAscarislumbricoidesandTaeniaspp.wereirradiatedwith

artiﬁcialwhitelight(0–0.5Wcm−2)withTetra-Py+_-Me_(5–30_␮M)

for15mininclearwaterand30mininwastewater.Therewasa

destructionofaround28%ofAscaris eggswith0.33Wcm−2 and

10␮MofTetra-Py+_-Me,_after₁₅_min_in_clear_water,_and_after₃₀_min

inwastewater.Theincreaseinthelightirradianceto0.5Wcm−2

caused 47% of destruction of Ascaris eggs in wastewater after

30minof irradiation.Thisincrease turnednegligiblethe

differ-enceinthesensitivityofAscariseggsinclearandwastewater.An

increaseintheoxygenationofthewastewaterandinthe

agita-tionprocessimprovedthephotosensitivityofTaeniaeggs.Infact,

theseeffects weremore evident when theconcentration of PS

wasalsoincreased.Theprocesswasoptimizedunderthe

follow-ingconditions:dissolvedoxygenwasmaintainedat7mgL−1,the

Tetra-Py+_-Me_{concentration}_at₃₀_␮M_and_the_egg_suspension_was

(12)

controlledbytheTetra-Py+_-Me_{concentration,}_the_irradiation_time

andthelightintensity.Also,thedissolvedoxygenconcentration,

waterqualityand thetypeofeggscaninﬂuencethesensitivity

ofhelmintheggstophotosensitization. Ascariseggswerefound

tobemoresensitivetophotosensitizationthanTaeniaeggs.The

sameauthorsalsousedTetra-Py+_-Me_in_several_{concentration}_(1.0,

5.0and10.0␮M)andsunlightirradiation(1235mWcm−2)upto

240mintoinactivatefecalcoliformsandfecalstreptococcion

sec-ondarywastewatersamples.After60min,at5.0and10.0_␮M,a

decreaseof2.94and2.4logsinfecalbacteriacountswasobserved,

respectively.After240min,atotalcellsurvivalreduction(>4.0log

units)wasachievedwithbothconcentrations.The5.0␮M

concen-trationwasconsideredtobemoresuitabletoreducefecalcoliforms

inwastewatersinceitallowsobtainingagoodtreatmentyieldand

itismoreeconomic[37].Thesuspended solids(turbidity)were

themostinﬂuentialsolutionparameterontheefﬁciencyofthe

photochemicalprocess.Turbidityreduceslightpenetration,which

reducesthePSexcitationandtheabsorptionbythehelmintheggs

[36].Thedecreaseinlogcountsoffecalcoliformswas≈1.0after1h

ofphototreatmentby5␮MTetra-Py+_-Me_when_suspended_solids

reached50mgL−1[37].

Albeittheseresultswerepromising,thepracticalapplication

ofphotodynamictreatmenttodisinfectmicrobiologicallypolluted

watersdependsonmanyfactors:theremovalofthePSafter

photo-dynamictreatmenttoavoidthereleaseofPStotheenvironment;

theuseofphotostablePS(i.e.,PSwhichdonotbleachunder

irradi-ation);theimpactofthisprocedureonthestructureofthenatural

non-pathogenicmicrobialcommunities;thetoxicityofthePSto

aquatic organisms at doses which inducemarked mortality on

microbialpathogens;theeffectofphysicalandchemical

proper-tiesofenvironmentalwaters;andthepossibilityofusingsunlight

aslightsource[28,32].

Ina pioneeringwork,Bonnettet al.[72] incorporated

meso-tetraarylporphyrins with amino and hydroxy substituents and

also a tetra sulfonated zinc phthalocyanine (ZnPcS4) into

chi-tosanmembranesforthespeciﬁcpurposeof waterdisinfection.

Modelreactorsforalarge-scalewater-ﬂowsystemweredesigned:

a static photoreactorsystem with7mL of bacterialsuspension

(3500cellsmL−1) and a circulating water photoreactor system

with 455mL of bacterial suspension (105_CFU _– _colony

form-ingunits–permL),representingthesigniﬁcantlevelsofwater

contamination. After 30min of irradiation with white light,

the chitosan membrane containing the

5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TetraTPPNH2) caused a reduction to

1300CFUmL−1.However,themembranepreparedwithZnPcS4,

wasmuch moreeffectiveandwasabletocompletelyinactivate

the bacteria after 30min. When the more efﬁcient membrane

wasstoredinthedarkforninemonths,thephotodynamicaction

wasstill detectable demonstrating itsthermodynamic stability.

Withthatmodelsystem,thephotoinactivationwithimmobilized

PScanbeusedtolowermicrobiallevelsinwater ﬂowsystems

[72].

Thebactericidal effectof silicagel-supported antimony

por-phyrin complexSbTPP/SiO2,under visible-lightirradiation over

LegionellapneumophilahasbeenreportedbythegroupofYasuda

etal.[97,98].Laboratoryandenvironmentalﬁeldexperimentsin

acoolingtowerof800Lcapacitywerecarriedoutusing

ﬂuores-centlampsaslightsource,andalsoinapublicfountainﬁlledwith

13m3_of_water_and_sunlight_irradiation._In_vitro,_the_inactivation

ofL.pneumophilawithSbTPP/p-SiO2(10mg)reducedthesurvival

rateto0.6%after60minofirradiation.Inthecoolingtower,after10

days,theconcentrationsofLegionellawerereducedtothedetection

limit,andtheselevelswerekeptuntiltheirradiationhadﬁnished.

Inthepublicfountain,thebacterialconcentrationswerereducedto

thedetectionlimit12daysaftertheSbTPP/SiO2catalysthadbeen

installedinthefountain.Thebacterialconcentrationswerekeptat

<30CFU100mL−1 for3monthsuntiltheremovalofthecatalyst

fromthefountain[97,98].

Pyrrolidine-fused chlorin derivatives (TPFPC) showed, after

immobilizedoneithersilicagelorMerriﬁeldresin-basedmaterial,

signiﬁcantactivityagainstEscherichiacoli(3.0logreductions)with

4.0mWcm−2after180min.Thephotostabilityofthematerialsand

theirpreservedactivityafter3successivecycleswasenvisagedas

apromisingoptionforwaterdisinfection[90,92].

Since 2004, our research group has developed a

broad-spectrumofPS,namelycationicporphyrins,whichcanefﬁciently

inactivate bacteria [18,71], bacterial endospores [135], viruses

[17,35,136]andfungi[105].Oneofthemosteffectivecompounds,

atricationicporphyrinwithapentaﬂuorophenylgroup

(5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentaﬂuorophenyl)porphyrin

tri-iodide,Tri-Py+_-Me-PF),_has_been_tested_on_bacteria_and_viruses

tochecktheviabilityrecoveryandresistancedevelopmentafter

repeatedincomplete photoinactivationcycles. Thebacteria and

virusesthatwereinactivatedtothedetectionlimitwithTri-Py+

-Me-PFdidnotrecoverviabilityafteroneweekandtheresistance

isnotenhancedafter10sub-lethalphotosensitizationtreatments

[38,41].Attheinitialstageofourwork,porphyrinswereusedto

photoinactivatefecalcoliformsandfecalenterococciin

wastewa-tersamplesfromasecondary-treatedsewageplant[93].Twoof

thecationic porphyrins used(Tri-Py+_-Me-PF_and _Tetra-Py+_-Me)

inactivated94–99.8%ofthefecalcoliformsat5␮Muponwhite

lightatlowirradiance(4mWcm−2)after270minofirradiation,

demonstratinghighefﬁciency.Inthisstudy,tworapidmonitoring

methods were used tooversee the bacterialphotoinactivation:

galactosidase activity as an indicator of the presence of fecal

coliformsand leucine incorporation asan indicatorof bacterial

activity.Theadvantages ofusingthesemethodsaretheireasier

andfasterperformanceagainstthedeterminationofCFU,giving

anexcellentrelationwithfecalindicatorsabundance[93].From

these ﬁrststudies, theexperimentalconditions were

standard-ized for testing the antimicrobial photoinactivation in vitro of

ourporphyrins infurtherstudies:cellculturesrangedfrom107

to 108_CFU_mL−1_, _porphyrin _{concentrations} _ranged _from _0.5 _to

5.0␮M, whitelight irradiationhad anirradiance of4mWcm−2

andthemaximumexposuretimewas270min(64.8Jcm−2).The

incubationtimeoftheorganismswiththeporphyrinswas10min

in the dark, previously to irradiation, in accordance with the

literature[14].Speciﬁcconditionsbeyondthesewillbedescribed

onthetext.

Later, seven synthetic cationic meso-substituted porphyrins

with one to four charges were tested on Enterococcus faecalis

and E. coli (107_CFU_mL−1_). _The _results _showed _that _the

tri-(Tri-Py+_-Me-PF_and_Tri-Py+_-Me-CO

2Me)andthetetra-cationicPS

(Tetra-Py+_-Me)_at_5.0_␮M_were_the_most_efﬁcient_ones,_reducing

E.colisurvivalby7logCFUmL−1after90minwithTri-Py+_-Me-PF

andTri-Py+_-Me-CO

2Meandafter270minwithTetra-Py+-Me[18].

The completephotoinactivation of bacteriawithlow light

irra-diance(4mWcm−2)suggeststhat PDIoffecalbacteriacanbea

possibilityforwastewaterdisinfectionundernaturallight

condi-tions.

Itwasalsoreportedthephotoinactivationofarecombinant

bio-luminescentE.colistrain whoselight emissiondecreased more

than4logwiththethreeporphyrinsused(Tetra-Py+_-Me,_Tri-Py+

-Me-PFandTri-Py+_-Me-CO

2Me).Theseresultswereachievedboth

withartiﬁcialwhitelight(4.0mWcm−2,64.8Jcm−2)andwith

sun-light(≈62mWcm−2,1004.4Jcm−2)after90–270minwith5.0␮M

ofPSbutTri-Py+_-Me-PF_was_the_most_efﬁcient_compound_[71]_._The

sameseriesofcationicporphyrinswerealsotestedon

bacterio-phagesisolatedfromwastewater,usingwhitelightand5.0␮Mof

porphyrin[17].Again,thetetra-andtricationicderivatives

inac-tivatedtheT4-likephagetothelimitsofdetection(reductionof

(13)

As nanotechnology emerges as an opportunity for water

treatmentpurposes [131,133,137,138], newmaterialsbased on

magnetic nanoparticles with different porphyrins covalently

immobilizedhave beenrecently developed, inorder tobe

eas-ilyremovedfromthewatermatrix,forsubsequentreuse[18,70].

Threedifferenthybridshavebeenpreparedandtestedin

micro-cosm conditions, being the multi-charged nanomagnet hybrid

basedonTri-Py+_-Me-PF_quite_effective_against_{Gram-positive}_and

Gram-negativefecalbacteria(5logdecreaseforE.faecalisandE.coli

at20␮MofPS)whenusingwhitelightirradiation(64.8Jcm−2).

Thishybridalsopresentedantiviralactivity,inactivatingthe

T4-likebacteriophage tothedetectionlimit(≈7logofinactivation)

[70].

In order to use this approach in operative ﬁeld conditions,

the multi-charged nanomagnet-Tri-Py+_-Me-PF _hybrid _has _been

recentlytested[111].Preliminaryresultsshowedthatthishybrid

iseffectiveinAllivibrioﬁscheriphotoinactivationin,atleast,a

six-cyclereuse,causingacumulativebacterialreductionhigherthan

37logunderlowlightirradiance(4.0mWcm−2).

Also,Tri-Py+_-C

14-Py+-Mehasbeenencapsulatedwithinsilica

microparticlesformingaconjugatewithca.0.9␮mdiameter.The

conjugateloadedwith12_␮MC14addedtoawatersample

contam-inatedwithmethicillin-resistantS.aureus(MRSA)(108_cells_mL−1_),

promotedatightassociationofthebacterialcellswiththesilica

microparticle–porphyrinsystem,andfurtherirradiationwith

vis-iblelight(30min,100mWcm−2)causeda3logreductioninthe

survivalofMRSAcellsinthewatersample.Theconjugateshowed

tobestableinaqueousmediumforatleast3months,easily

recov-eredbyﬁltrationof theaqueoussuspension andkeptthehigh

antibacterialphoto-activitywhenreused[91].

Another example of recovery and reuse of immobilized

porphyrin used protoporphyrin IX (PPIX) attached to

acid-functionalizedmulti-walledcarbonnanotubes(NT-PPIX)aspotent

antiviralagentstobeusedonsurfacedisinfectionasacoatingor

inwatertreatment[96].InﬂuenzavirusstrainX-31,A/Aichi/2/68

(H3N2)wasirradiatedwithvisiblelightand1mgmL−1ofNT-PPIX

upto90min.Thistreatmentcausedmorethana250-fold

reduc-tionintheeffectiveinfectiousviraldoseaftera30minexposure

tolight.Thepercentageofcellsthatcouldbeinfectedby200ng

ofviruswasonlyabout1%.Apre-exposureofNT-PPIXfor90min

showedapartiallossofphotoactivity,butitseffectonthevirus

wasstillequivalenttoareductionintheinfectiousviraldoseby

almost50-fold.ReusabilitystudiesofNT-PPIXshowedtobe

depen-dentonphotobleachingoftheporphyrinmoietyandontheyieldof

recoveryofNT-PPIXaftereachuse.Besidesbeingeffective

bacte-ricidalagentsaspreviouslyprovedonStaphylococcusaureus[107],

theseconjugatesmaybeappliedasreusableantiviralsin

wastew-atertreatment,astheycanbeeasilyrecoveredwithoutleavingany

toxicby-products[96].

Althoughthereisatremendousneedforscientiﬁcknowledge

ondisinfectionofhospitalwastewater,thereisonlyonereport,

fromourgroup,ontheuseofphotodynamic treatmentonthis

typeofefﬂuent.Theefﬁciencyofphotoinactivationonfour

multi-drugresistantisolatedstrainsofE.coli,Pseudomonasaeruginosa,S.

aureusandAcinetobacterbaumanniiwasevaluatedinbuffered

solu-tionandinhospitalwastewater,using5.0␮MofTetra-Py+_-Me_and

whitelight(64.8Jcm−2)[139].Theresultsshowedanefﬁcient

inac-tivationofmultidrug-resistant(MDR)bacteriainbufferedsolution

(reductionof6–8logCFUmL−1)andinthewastewater,inwhich

thephotoinactivationofthefourbacteriawasalsoeffectiveand

thedecreaseinbacterialnumberoccurredevensooner.This

dis-similaritywasassignedtodissolvedcompoundsin thehospital

wastewater,suchasantibiotics.

Aquacultureactivityisincreasingworldwide,motivatedbythe

progressivereductionofnaturalﬁshstocks.Thisactivitysuffers,

however,substantialﬁnanciallossesresultingfromﬁshinfections

bypathogenicmicroorganisms.Themisuse ofawide varietyof

antibioticsinaquaculturehasraisedseveralproblemssuchasthe

emergenceofMDRbacteriainaquacultureenvironments;therise

in antibioticresistance in ﬁshpathogens; the transferof these

resistancedeterminantstobacteriaoflandanimalsandtohuman

pathogens;andmodiﬁcationsofthebacterialﬂorabothinwater

columnandinsediments[140].Moreover,althoughcommercial

vaccinesagainstthemostcommonpathogensareavailableforﬁsh,

vaccinationisnotanoptionforﬁshlarvae,oneoftheﬁshlifestages

mostpronetomicrobialinfection,asitisunfeasibletohandlelarge

numbersofthesesmall-sizedandfrailorganisms.Furthermore,ﬁsh

larvaedonothavetheabilitytodevelopspeciﬁcimmunity[141].

Consequently,PDIcanbeanalternativeapproachtotreatﬁsh

lar-vae.Thetreatmentcanbeappliedasapreventiveapproachagainst

bacterialinfectionsduringlarvaeproduction,beforereleasingthem

intheaquaculturetanks,therebyimprovingtheoverallproduction

ofadultﬁshandthesustainabilityofﬁshfarming.

As a consequence, strategies to control ﬁsh pathogens are

neededandPDIcanbeanoptiontotreatdiseasesandtoprevent

thedevelopmentof antibioticresistancebypathogenicbacteria

[142].AsthePScouldbedegradedandusedinalow

concentra-tion,itwouldbeinnocuoustoanimals,toﬁshconsumersandto

theenvironmentHowever,theideaisnottousethePSdilutedor

dispersedintheaquaculturewater,butinstead,useitimmobilized

insolidsupportsinorderthatitcouldberecoveredandreused

suc-cessively,withoutbeingdiscarded.Aquaculturewatertreatedwith

immobilizedPSundersunlightmakesthisapproachattractive,cost

effectiveandeco-friendly.

The photodynamic disinfection of water from ﬁsh farms as

wellasthe preventionand treatmentoflocalized fungal

infec-tions(saprolegniosis)inﬁsh,withporphyrinsasphotosensitizing

agents, was presented in 2006, demonstrating the inactivation

invitroofpathogenicfungiandbacteria,andthephoto-treatment

ofspontaneouslyorartiﬁciallyinfectedﬁshinapilotaquaculture

pond [81]. The PS chosen were Tetra-Py+_-Me _and _Tri-Py+_-C

14

-Py+_-Me _(0.05–10_␮M). _The_treatment _of _infected _rainbow_trout

(Oncorhynchusmykiss)consistedintwoprotocols:preventiveand

curative(Fig.4).In thepreventive protocol,artiﬁcially infected

ﬁshin1000Lcapacitytanksweredarkincubatedfor10minwith

0.2␮M ofTri-Py+_-C

14-Py+-Meand werenot irradiated, orwere

incubatedfor 10min with0.44␮M of Tetra-Py+_-Me _and

irradi-atedfor1hwithwhitelight(50mWcm−2).Theincubationand

irradiationtreatmentwasrepeateddaily,fortenconsecutivedays,

startingfromtheﬁrstdayaftertheinfection.Inthecurative

proto-col,infectedﬁshweredarkincubatedwith0.44␮MTetra-Py+_-Me

for10mininan80Lpool,irradiatedfor1handmovedtoa1000L

tank(treatmentrepeateddailyforsixconsecutivedays);ordark

incubatedwith0.4␮MofTri-Py+_-C

14-Py+-Mefor24hina150L

tank,notirradiatedandthenmovedtoa1000Ltank[81].

ExperimentswithSaprolegnia(≈108_zoospores_mL−1_),_indicated

thatTri-Py+_-C

14-Py+-Mecausedadecreaseofthesurvivalofthe

fungal cells by about 6 logs, after phototreatment with 10␮M

[81]. The preventive protocol determined a reduction of the

infectedpercentageto10%and13%,respectively,afteroneweek,

withirradiated Tetra-Py+_-Me _and _unirradiated _Tri-Py+_-C

14-Py+

-Me,respectively.Aftertwoweeks,alltheinfectedindividualsfrom

alltheexperimentalgroups recoveredfromtheinfection.With

thecurativeprotocol,acompleteremissionoftheinfectionwas

inducedwithinoneweek,followedbythecompletehealingofthe

ulceratedlesion.Gradualphotobleachingofbothporphyrinswas

observedbutphotodegradationproductswerenottoxic[81].

Inthecaseoftreatinginfectedﬁshbythisapproach,eitherin

apreventiveorcurativeway,thePSwasaddedtothewatertank

followedbyirradiationwithartiﬁcialwhitelight[81](Fig.4).In

thatcase,theporphyrinswereabletointeractwiththepathogen

(14)

inactivationorpreventtheirproliferation.Theinduced-infections

bySaprolegniasp.introutswereexternal,onthedorsalregion,thus

theinfectionwastreatedbyexposingthelesionintheﬁshtowater

addedwithporphyrinandafteraperiodofirradiation.

Foraquaculturewater disinfectionusingPDI,thePSmaybe

addedtowater,inasolidmembraneordispersedinparticlesto

disinfectthismatrixbeforebeingincontactwithﬁsh.

Microbiolog-icallycontaminatedwaterwouldbedecontaminatedpriortobeing

deliveredtotheﬁshtankculture.However,treatingﬁshdiseases

throughPDIisanotherconcernthathasnotbeenobjectofenough

scientiﬁcresearch.

Studiescarriedoutinourlaboratoryindicatedthatnine

bacte-rialspecies(Vibrioanguillarum,V.parahaemolyticus,Photobacterium

damselaesubsp.damselae,P.damselaesubsp.piscicida,Aeromonas

salmonicida,E.coli,Pseudomonassp.,S.aureusandE.faecalis)

iso-lated from ﬁsh-farming plant water are effectively inactivated

(upto7logunits)withthetricationic porphyrinTri-Py+_-Me-PF

at5.0␮M,underalow lightirradiance(4.0mWcm−2)[49].The

cultivablefractionoftheheterotrophicbacteriaofthesame

aqua-cultureplant,includingpathogenicandnon-pathogenicbacteria,

wasalso inactivated byPDI, but the efﬁciencyof the

inactiva-tion varied during thesampling period. The seasonal variation

ofphotoinactivationefﬁciencycanbeduetodifferencesin

bac-terial community structure but also due to variations of the

waterphysico-chemicalproperties.Theresultsclearlyshowthat

it ismore difﬁculttophotoinactivate thecomplex natural

bac-terialcommunities of aquaculturewaters thanpurecultures of

bacteria isolated from these waters [49]. As PDI is not

selec-tiveforpathogenicmicroorganisms,thenon-pathogenicmicrobial

communityof aquaculturewaterscanalsobeaffected.As

non-pathogenic bacteria have an important ecological role in the

biogeochemical cycles in aquaculture waters, namely in

semi-intensivesystems,acarefulevaluationoftheenvironmentalimpact

mustbeconductedbeforePDIimplementationinthesesystems.

Preliminaryresultsfromourgroupshowedthatnotallbacterial

populationsareaffectedbyPDI.Inaquaculturewatertreatedwith

Tri-Py+_-Me-PF_and_exposed _to_light, _a_reduction _on_the_number

ofbacterialgenotypesrelativelytothenon-treatedwatersamples

hasbeenobserved,indicatingthatdominantbacterialpopulations

wereaffectedbyPDI.However,bacterialpopulationisalsoaffected

bysimplelightexposure[49].

Theeffectofphysico-chemicalpropertiesofaquaculturewaters

onPDIefﬁciencyhasalsobeenevaluated[110].ThePDIassayswere

designedhavingintoaccounttheannualvariabilityofpH(6.5–7.5),

temperature (13–22◦C), salinity (10–35gL−1)and oxygen

con-centration(2–6mgL−1)valuesintheﬁshfarmswherethewater

wascollected.TomonitorthePDIkinetics,thebioluminescenceof

A.ﬁscheriwasmeasured.ThevariationsinpH,temperature,

salin-ityandO2didnotsigniﬁcantlyaffectthePDIofV.ﬁscheri(≈7log

reductioninallconditions)with5.0␮MofTri-Py+_-Me-PF_under

whitelight(4.0mWcm−2).Thesuspendedsolidsinthe

aquacul-turewaterreducedtheefﬁciencyofPDIinrelationtoclearaqueous

solutions(bufferedsolution).Ontheotherside,agoodresponse

wasobtainedinaquaculturewaterbyincreasingthePS

concentra-tionto20␮M.ThekineticsofthephotoinactivationofA.ﬁscheri

inaquaculturewater usingtwodifferentlightsources(artiﬁcial

whitelight and solarlight)wasalsotested. Theinactivation of

V.ﬁscheriundersolarlight(40mWcm−2)wascomparedtothat

obtainedwithartiﬁcialwhitelight,usingthesametotallightdose.

Theresultsobtainedwith20␮Mofporphyrinshowedthat,using

thesametotallightdose(64.8Jcm−2),bothlightsources

inacti-vateA. ﬁscheritothedetectionlimit.Asit isintendedthat this

technologywillbeusedinrealaquaculturecontext,usingsolar

irradiationaslightsourceisexpectedtomakethiswater

disinfec-tionapproacheconomicallysustainableintermsofenergydemand

[110].

Toovercometheentericsepticemiaofcatﬁsh,columnaris

dis-easeandthepresenceofodor-producingcyanobacteriainpondsof

catﬁsh(Ictaluruspunctatus)production,theantibacterialand

algi-cidalactivityofTri-Py+_-C

12-Py+-Mehasbeenevaluatedontheﬁsh

pathogensEdwardsiellaictaluriandFlavobacteriumcolumnareand

onS.aureusforcomparison[69].ThesamePSwasalsousedonthe

odor-producingplanktoniccyanobacteriumPlanktothrixperornata

andonarepresentativegreenalgaSelenastrumcapricornutum.

Tri-Py+_-C

12-Py+-Mewasusedinconcentrationsrangingfrom0.01to

1000␮Mandthesampleswereirradiatedwithﬂuorescentlamps

ataphotonﬂuxdensityof16–30␮Em−2s−1.Minimalinhibitory

concentrationswereobtainedasfollows:9.8mgL−1forE.ictaluri,

0.5–1.0mgL−1 for F.columnare and0.1mgL−1 forS.aureus.The

growthinhibitionafter96hirradiationwith0.07and0.2mgL−1

ofC12porphyrin,inhibited50%ofbothP.perornataandS.

capri-cornutum,respectively(totalinhibitionwasobservedat1.0mgL−1

inbothcases).Whileinvivotestsorothertoxicitytestwerenot

reported, thepreliminary ﬁndings showed thebroad spectrum

activityofTri-Py+_-C

12-Py+-Meandsuggestedapplicationsinclude

sanitizingemptytanksbeforerestockingandtreatmentofother

aquaculturesystemsdealingwithsimilardiseaseoutbreaks[69].

Tri-Py+_-C

12-Py+-Me, already mentioned as an effective

pho-toactivatedantimicrobialagentforaquaculture[69]and

disease-transmitting insect vectors [50], has also been tested on the

protozoanCiliophoraColpodainﬂataandTetrahymenathermophile

andontheCrustaceaBranchiopodaArtemiafranciscanaand

Daph-nia magna, which are used in routine toxicity assessment in

freshwater ecosystems[52]. Followingincubation withTri-Py+

-C12-Py+-Me for 60min (0.1–10␮M range) the organisms were

irradiatedwithvisiblelight(10mWcm−2)for60minaswell.

Tri-Py+_-C

12-Py+-Mecausedagrowthinhibition≥90%againstC.inﬂata

cystsortrophozoiteswith0.3–0.6␮M,3hafter irradiationwas

ended.Inturn,T.thermophilavegetativecellsrequired3␮Mfora

50%inhibitionofgrowth,46hafterirradiation.Thecomplete

inacti-vationofD.magnawasachievedwith0.6_␮MofTri-Py+_-C

12-Py+-Me

while in A. franciscanaphotosensitivitywasnot detectedup to

10␮M.Fluorescencemicroscopyanalysesclearlyrevealeda

dam-agedmorphologyinducedoncystsofC.inﬂataby1␮MofPSand

ontrophozoitesofC.inﬂataby0.6␮M.T.termophilaalsorevealed

damagewith6␮MofTri-Py+_-C

12-Py+-Me.D.magnaaccumulated

thePSinthedigestivetractandexoskeletonand A.fransciscana

revealedaccumulationespecially ontheexoskeleton.The

resis-tanceofA.franciscanatothephototreatmentwasexplainedbythe

verylowamountoftheingestedPSalongwithitsabilitytoadaptto

extremeconditions.Theseﬁndingsemphasizedtheneedfor

care-fullytailoredirradiationprotocols,takingintoaccountthenature

ofthespeciﬁcwaterbasin,particularlyinwhatreferstoitsbiotic

characteristics[52].

Recently, chlorophyllin-mediated PDI has been suggested

as a new promising treatment to control ichthyophthiriosis, a

whitespot-causingdiseaseinmanyfreshwaterﬁshspeciesbythe

protozoanparasiteIchthyophthiriusmulftiﬁliis.Differentlifestages

oftheparasiteweretested:trophonts(encystedstageinthehost)

and tomites (motile, infective stage). Samples were incubated

60mininthedarkwith0.5–10␮gmL−1,followedbyirradiation

withsimulatedsunlight(PAR149.66Wm−2,UV-A32.67Wm−2,

andUV-B0.77Wm−2)for30min.Thephotodynamiceffectonthe

cellswasevaluatedbyﬂuorescencemicroscopeafterstainingwith

theﬂuorescentdyeacridineorange.Transmittedlightmicroscopy

showed that chlorophyllin completely ﬁlled the trophont and

destroyedthenucleusandthecellwall.TheLC50value,calculated

fortrophontsofI.multiﬁliis,was0.67␮gmL−1 withamaximum

mortality of about 10% observed in dark conditions. Even at a

chlorophyllin concentrationof 2_␮gmL−1 in themedium, 100%

ofthetomitesweredead.Theresultsoftheinvitroexperiments