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Journal of Photochemistry and Photobiology C Photochemistry Reviews 22 (2015) 34ndash57

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology CPhotochemistry Reviews

jo u r nal homep age wwwelsev ier com locate jphotochemrev

eview

otential applications of porphyrins in photodynamic inactivationeyond the medical scope

liana Alvesa Maria AF Faustinob Maria GPMS Nevesb Acircngela Cunhaaelena Nadaisc Adelaide Almeidaalowast

Department of Biology and CESAM University of Aveiro Campus de Santiago 3810-193 Aveiro PortugalDepartment of Chemistry and QOPNA University of Aveiro Campus de Santiago 3810-193 Aveiro PortugalDepartment of Environment and Planning and CESAM University of Aveiro Campus de Santiago 3810-193 Aveiro Portugal

r t i c l e i n f o

rticle historyeceived 4 February 2014eceived in revised form 29 August 2014ccepted 3 September 2014vailable online 21 September 2014

eywordsorphyrinhotosensitizerhotodynamic inactivationisinfectionanomaterialself-cleaning materials

a b s t r a c t

Although the discovery of light-activated antimicrobial agents had been reported in the 1900s only morerecently research work has been developed toward the use of photodynamic process as an alternativeto more conventional methods of inactivation of micro(organisms) The photoprocess causes cell deaththrough irreversible oxidative damage by reactive oxygen species produced by the interaction betweena photosensitizing compound and a light source

With great emphasis on the environmental area photodynamic inactivation (PDI) has been tested ininsect eradication and in water disinfection Lately other studies have been carried out concerning itspossible use in aquaculture waters or to the control of food-borne pathogens Other potential applicationsof PDI in household industrial and hospital settings have been considered

In the last decade scientific research in this area has gained importance not only due to great develop-ments in the field of materials chemistry but also because of the serious problem of the increasing numberof bacterial species resistant to common antibiotics In fact the design of antimicrobial surfaces or self-

cleaning materials is a very appealing idea from the economic social and public health standpoints ThusPDI of micro(organisms) represents a promising alternative

In this review the efforts made in the last decade in the investigation of PDI of (micro)organismswith potential applications beyond the medical field will be discussed focusing on porphyrins free orimmobilized on solid supports as photosensitizing agents

copy 2014 Elsevier BV All rights reserved

ontents

1 Introduction 352 Applications on the environment water and foodstuff 36

21 Insect pest elimination 3622 Water disinfection 4423 Elimination of food-borne pathogens 48

3 Applications for domestic industrial and healthcare settings 5031 Porphyrin-embedded fabric 5032 Porphyrin-embedded paper 5133 Porphyrins immobilized in other support materials 51

4 Conclusions Acknowledgements References

lowast Corresponding author Tel +351 234 370 784E-mail address aalmeidauapt (A Almeida)

ttpdxdoiorg101016jjphotochemrev201409003389-5567copy 2014 Elsevier BV All rights reserved

53 53

53

tobiol

E Alves et al Journal of Photochemistry and Pho

Eliana Alves was born in Porto (Portugal) She receivedher MSc degree in 2007 and her PhD degree in Biol-ogy in 2013 both from the Department of Biology andCESAM of the University of Aveiro (Portugal) During sevenyears her research work was totally dedicated to Photobi-ology namely to microbial photodynamic therapy usingcationic porphyrins as photosensitizers At the momentshe is a Postdoctoral Research Fellow at the Departmentof Chemistry and QOPNA of the University of Aveiro whereshe is acquiring expertise in lipidomics an expanding fieldof lipid analysis through chromatographic and mass spec-trometry techniques

Maria Amparo F Faustino received her doctoraldegree in Chemistry from University of Aveiro in1999 She is currently working as Assistant Profes-sor in the Department of Chemistry University ofAveiro Her current area of research includes synthe-sis and transformation of tetrapyrrolic derivatives andtheir applications mainly environmental applicationshttporcidorg0000-0003-4423-3802

Maria da Grac a PMS Neves is Associated Professor withHabilitation at the Department of Chemistry of the Univer-sity of Aveiro (UA) She obtained her Habilitation and PhDdegree from UA her MSc degree from UMIST Manch-ester (Great Britain) and her BSc degree in Chemistryby University of Lourenc o Marques (Mozambique) Herresearch interests are centered on the synthesis func-tionalization and potential applications of tetrapyrrolicmacrocycles like porphyrins corroles and phthalocya-nines httpsorcidorg0000-0002-7953-8166

Acircngela Cunha is Assistant Professor at the Department ofBiology and researcher of the Center for Environmentaland Marine Studies (CESAM) of the University of Aveiro(Portugal) She received her PhD degree in Biology in2001 at the University of Aveiro and has been workingon environmental microbiology and microbial ecology ofaquatic environments Her recent interests are the studyof the effect of biosurfactants on the development ofmicrobial biofilms and on of photodynamic approaches forthe prevention and control of microorganisms with a par-ticular focus on resistant structures such as endosporesand polimicrobial biofilms

M Helena G A G Nadais is Assistant Professor at theDepartment of Environment and Planning in the Univer-sity of Aveiro (UA) She is a Chemical Engineer holding aMSc in Chemical Engineering (1993) from Instituto Supe-rior Tecnico Technical University of Lisbon Portugal anda PhD (2002) in Environmental Science from the UA Herresearch interests are centered on water treatment andon material and energetic valorization of wastewaters andwastes She has more than 60 publications She has partic-ipated whether as team member or as main investigator invarious scientific research projects and recently submit-ted a patent application She is full member of the researchcenter CESAM (wwwcesampt)

Adelaide Almeida is Assistant Professor at the Depart-ment of Biology from the University of Aveiro (Portugal)where she got her PhD degree in 2001 She is an inte-grated member of the Associated Laboratory Centre forEnvironmental and Marine Sciences (CESAM) In the lastyears she has been involved in the development and

application of alternative methods to the use of antibi-otics such as photodynamic therapy and phage therapyShe has published in these fields and her publications canbe found in httpwwwcesamuaptadelaidealmeida

ogy C Photochemistry Reviews 22 (2015) 34ndash57 35

1 Introduction

Photodynamic therapy refers to the use of a light source (visiblelight of an appropriate wavelength) an oxidizing agent (molecularoxygen O2) and an intermediary agent (named photosensitizer PS)able to absorb and transfer the energy of the light source to molec-ular oxygen leading to the formation of highly cytotoxic species(singlet oxygen [1O2] hydrogen peroxide [H2O2] andor free radi-cals such as superoxide [O2

minusbull] and hydroxyl radical [HObull]) causinga multi-targeted damage and destruction of living tissues [12]The generation of these reactive oxygen species (ROS) can occurvia two mechanisms or pathways known as type I and type IIwhich require the presence of O2 (Fig 1) In the presence of light(h) the photosensitizer in the singlet ground state absorbs a pho-ton affording the excited singlet state Then it can lose energy byreturning to the singlet ground state with fluorescence emission(F) or through an intersystem crossing (ISC) process it can be con-verted in the long-lived triplet state This excited triplet-state PScan decay to ground state by phosphorescence emission (P) or canreact with a substrate namely an electron donor molecule In thiscase the formation of radical ions can occur giving rise to radicalions which react with ground state oxygen (3O2) originating ROS(type I mechanism) Alternatively the excited triplet-state PS cantransfer energy directly to molecular oxygen affording the excitedsinglet state (1O2) (type II mechanism) Both photoprocesses mayoccur simultaneously but type II is in general the predominant oneThe cytotoxic species can cause irreversible damage to proteinsnucleic acids and lipids [34]

The advantage of being a process without a specific cell targetrenders photodynamic inactivation (PDI) effective in the oxidationof different biomolecules with the consequent destruction of sev-eral cell types In fact this methodology has a broad spectrum ofactivity and using the same PS is able to destroy human cells[1] viruses [5] bacteria [6] molds [7] yeasts [8] protozoa [9]helminths [10] and insects [11]

Moreover the ability to structurally tailor the PS as well as tosuccessfully link it to other molecules with a high degree of speci-ficity (eg antibodies enzymes nucleic acids) or to solid supportsgives this therapy a multiplicity of clinical and non-clinical appli-cations

The discovery that positively charged PS could effectivelyinactivate Gram-negative bacteria without the addition of perme-abilizing agents [1213] brought a new impetus to the investigationon the PDI of microorganisms as a new therapeutic modal-ity

The difference in susceptibility between the two types of bac-teria Gram-negative and Gram-positive is explained on the basisof the structural features of their cell wall (Fig 2) Gram-positivebacteria have a cell wall composed of lipoteichoic and teichoicacids organized in multiple layers of peptidoglycan which confersa degree of porosity to bacteria so as to facilitate the anchoringand entry of PS into the cell [1415] In Gram-negative bacte-ria the presence of a complex outer membrane in the cell wallconsisting of phospholipids lipopolysaccharides lipoteichoic acidsand lipoproteins creates an impermeable barrier to antimicrobialagents [1415] The interaction between the cationic PS and theconstituents of the Gram-negative cell wall generates electrostaticinteractions that promote destabilization of the native organiza-tion of the wall allowing the binding and eventual entry of the PSmolecules into the cell [1415] In the case of fungi the cell wallcontains chitin glucans and lipoproteins that represent a barrierwith intermediate permeability in comparison to Gram-positive

and Gram-negative bacteria [16] With regard to viruses envelopedviruses are more easily inactivated than non-enveloped ones butsome studies show that non-enveloped viruses can also be effi-ciently inactivated by the phototoxic action of cationic PS [17]

36 E Alves et al Journal of Photochemistry and Photobiology C Photochemistry Reviews 22 (2015) 34ndash57

Singletgroun dstate PS

Sing letexcitedstate PS

Tripletexcitedstate PS Dioxygen

Substrate Reacve oxygen species

Singlet oxygen

Irr eversible damage onmajor cell compon ents

Type I mechanism

Type II mechanism

h ν

FISC

ICISC

ng pho

bn

ht(p(f

oteon

sTva

t[tgr

wufl(howFlvdrct

P

Fig 1 Schematic representation of the photoprocesses that can occur duri

eing the efficiency of their inactivation similar to that of Gram-egative bacteria [18]

In recent years the synthesis of new compounds for PDIas grown dramatically many of them with very good inactiva-ion results Several classes of PS such as phenothiazinium dyesmethylene blue toluidine blue O) naturally occurring PS (chloro-hylls psoralens perylenequinonoid pigments) tetrapyrrolesporphyrins phthalocyanines chlorins bacteriochlorins) andullerenes have been successfully tested [19ndash21] (Fig 3)

The group of porphyrin derivatives has been prominent notnly because it includes the first formulation approved for pho-odynamic therapy of cancer [22] but also in the perspective ofnvironmental applications considering that the use of naturallyccurring porphyrins or synthetic related analogs arises as an eco-omical eco-friendly and humananimal safe option [23]

Porphyrins are a class of heterocyclic aromatic compounds con-tituted by four subunits of pyrrole type linked by methynic bridgeshe presence of these compounds is ubiquitous in nature as part ofital biochemical processes such as oxygen transport (heme group)nd photosynthesis (chlorophylls) [24]

If photodynamic therapy is now an established procedure forhe treatment of certain non-oncological and oncological diseases222526] it is still not used for infection treatment [1527] In addi-ion the potential application of PDI to destroy (micro)organismsoes beyond the medical field with particular focus on the envi-onmental area [28ndash32]

In PDI studies related with non-clinical applications artificialhite light (halogen or xenon lamps) and sunlight have beensed in order to achieve organismsrsquo destruction The irradiance (oruence rate) can be given in W mminus2 in lux (lx) or in E mminus2 sminus1

with E standing for Einsteins in the former terminology whichas been replaced in the new terminology by M meaning ldquomolef lightrdquo) These light units can be interconverted in the followingay 1 lx asymp 95 times 10minus3 mW cmminus2 asymp 18 times 10minus3 M mminus2 sminus1 [33]

or example 100ndash150 lx may represent a shady room in naturalight 30000ndash40000 lx an overcast summerrsquos day and 100000 lx aery bright summerrsquos day [34] Since the experimental conditions

escribed in literature reports are quite different sometimes theesults cannot be directly compared The same light dose (J cmminus2)an be achieved by varying the light irradiance the irradiationime or both [21] However the effectiveness of the results may be

Fig 2 Schematic representation of the cellular envelope of G

todynamic inactivation ISC intersystem crossing IC internal conversion

different when using a high irradiance over a short time period or alow irradiance over a longer time even though the light dose is thesame in each case [2135] In general the PDI of microorganismsis more extensive when higher irradiance and longer treatmentduration are used [36] Increasing the duration of irradiation willimprove the yield of treatment compensating a low concentrationof PS or a less efficient PS type [37]

Due to its multi-target nature and therefore low probabilityof triggering the development of resistance in (micro)organisms[38ndash41] this therapy has been tested in various research areasas an alternative approach to actual methods to control insectpests water quality microbiological food quality and also in thedisinfection and sterilization of materials and surfaces in differ-ent contexts (industrial household and hospital) Furthermore theuse of this form of eradicating microbes or noxious organismsbecomes increasingly achievable in practice if one thinks that forcertain purposes the PS may be immobilized on inert solid sup-ports allowing their reuse and recycling making this technology ofbroad-spectrum activity economic sustainable durable and envi-ronmentally friendly To this extent with the great developmentof nanotechnology and materials chemistry several different sup-ports have been created to immobilize a series of PS designed forphotoinduced oxygenation reactions [42]

The aim of this review is to present the efforts made in the lastdecade in the investigation of PDI of (micro)organisms beyond themedical field using porphyrins as PS either free or immobilizedin solid supports All the organisms which have been used on PDIexperiments are listed in Table 1 The porphyrinic PS reported inthese studies are listed in Table 2

2 Applications on the environment water and foodstuff

21 Insect pest elimination

Alternative pesticides for pest and vector control have beenrequired for more than a decade Insecticide industry and vectorcontrol management face several problems nowadays devel-

opment of resistance in major vectors to common insecticidesabandonment of certain compounds for safety reasons and envi-ronmental and human health concerns about the use of many oldergeneration insecticides such as DDT economic factors and market

ram-positive (left) and Gram-negative bacteria (right)

E Alves et al Journal of Photochemistry and Photobiology C Photochemistry Reviews 22 (2015) 34ndash57 37

Table 1List of organisms used in non-clinical photodynamic inactivation experiments

Organism Organism type References

Acinetobacter baumannii Bacterium (Gn) [43]Acremonium spp Fungus (mold) [44]Acremonium strictum Fungus (mold) [45]Aedes aegypti Mosquito (dengue vector) [4647]Aedes caspius Mosquito [48]Aeromonas salmonicida Bacterium (Gn) [49]Alternaria alternata Fungus (mold) [45]Alternaria spp Fungus (mold) [44]Anopheles arabiensis Mosquito (malaria vector) [50]Anopheles gambiae Mosquito (malaria vector) [50]Anopheles sp Mosquito (malaria vector) [51]Artemia franciscana Crustacea (Branchiopoda) [52]Ascaris lumbricoides Helminth [36]Aspergillus flavus Fungus (mold) [7]Aspergillus spp Fungus (mold) [44]Bacillus cereus Bacterium (Gp) [53ndash58]Bactrocera oleae Fly (olive fly) [59]Baculovirus Enveloped DNA virus [60]Candida albicans Fungus (yeast) [61ndash63]Ceratitis capitata Fly (Mediterranean fluit fly) [5964]Chaoborus crystallinus Insect larvae (Diptera) [2365]Chaoborus sp Insect larvae (Diptera) [66]Colpoda inflata Protozoan (Ciliophora) [52]Culex pipiens Mosquito [6768]Culex quinquefasciatus Mosquito [46]Culex sp Mosquito [66]Cultivable bacteria from aquaculture water Bacteria [49]Daphnia sp Daphnia magna Crustacea (Branchiopoda) [5266]Edwardsiella ictaluri Bacterium (Gn) [69]Enterococcus faecalis Bacterium (Gp) [184970]Escherichia coli Bacterium (Gn) [1841496370ndash92]Fecal coliforms Bacterium (Gn) [3793]Fecal enterococci Bacterium (Gp) [3793]Flavobacterium columnare Bacterium (Gn) [69]Fusarium avenaceum Fungus (mold) [745]Fusarium culmorum Fungus (mold) [94]Fusarium poae Fungus (mold) [94]Fusarium spp Fungus (mold) [44]Ichthyophthirius mulftifiliis Protozoan [6595]Influenza virus strain X-31 AAichi268 (H3N2) Virus [96]Legionella pneumophila Bacterium (Gn) [9798]Liriomyza bryoniae Fly (leafminer fly) [99]Listeria monocytogenes Bacterium (Gp) [535780100ndash103]Mucor spp Fungus (mold) [44]Musca domestica Fly (house fly) [68]Mycelia sterilia Fungus (mold) [44]Mycobacterium smegmatis Bacterium (Acid-fast) [75]Parasarcophaga argyrostoma Fly (flesh fly) [104]Penicillium chrysogenum Fungus (mold) [105]Penicillium spp Fungus (mold) [44]Photobacterium damselae subsp damselae Bacterium (Gn) [49]Photobacterium damselae subsp piscicida Bacterium (Gn) [49]Planktothrix perornata Cyanobacterium [69]Polyomavirus Virus (Non-enveloped DNA virus) [60]Pseudomonas aeruginosa Bacterium (Gn) [4383]Pseudomonas sp Bacterium (Gn) [49]Rhizopus oryzae Fungus (mold) [745]Rhizopus spp Fungus (mold) [44]Saccharomyces cerevisiae Fungus (yeast) [8]Salmonella enterica Bacterium (Gn) [102106]Salmonella sp Bacterium (Gn) [80]Saprolegnia spp Fungus (mold) [81]Selenastrum capricornutum Green alga [69]Staphylococcus aureus Bacterium (Gp) [43496973ndash7577ndash83868791107108]Stomoxis calcitrans Fly (house fly) [109]T4-like phage Bacteriophage [173870]Taenia sp Helminth [36]Tetrahymena thermophila Protozoan (Ciliophora) [52]Trichothecium roseum Fungus (mold) [7]Ulocladium oudemansii Fungus (mold) [8]Vibrio anguillarum Bacterium (Gn) [49]Vibrio fischeri Bacterium (Gn) [110111]Vibrio parahaemolyticus Bacterium (Gn) [49]

Gn Gram-negative Gp Gram-positive


ers us

cairo

eifotAcma

ip[dcspAltsipRiA

Fig 3 Classes of natural and synthetic photosensitiz

onstraints lack of available safe and cost-effective larvicides forddition to drinking water depletion of safe and cost-effectivensecticides re-emergence of disease caused by the early efforts toeduce indoor residual insecticide application rates and increasef vector-borne disease transmission [112ndash114]

Therefore there is an urgent need for improved novel cost-ffective insecticides and insecticide treated materials long-lastingnsecticide formulations for such materials and sprays economiceasibility and commercialization analyses for pesticides devel-pment and most importantly providing solutions to overcomehe increasing problems of resistance to current insecticidesccordingly it has been suggested the development of insecti-ide combinations such as bi-treated nets and insecticide treatedaterials [112] or new active ingredients based on novel modes of

ction [115]The idea of using PS as photopesticides (or photoinsecticides)

s not new [116117] However the use of porphyrins for this pur-ose was only mentioned from the late 1980s of the last century118ndash121] Since then the use of natural or synthetic porphyrinerivatives has been increasingly exploited to control and eradi-ate various types of insects including pest flies capable of inducingignificant damage to agricultural crops and mosquitoes vectors ofathogens responsible for malaria (Anopheles) yellow fever (Culexedes) dengue fever (Aedes) and encephalitis (Aedes Culex Anophe-

es) [46ndash485066ndash68] Nowadays there is a global distribution ofhese organisms In the case of Aedes aegypti for example itsporadic presence has been recognized in mid-latitudes such asn Britain France Italy Spain Malta Portugal and other Euro-ean countries [122123] Recently in Madeira island (Autonomous

egion of Madeira) Portugal there was a dengue outbreak starting

n October 2012 with 2170 cases of dengue fever notified until earlypril 2013 In mainland Portugal 11 cases have been reported and

ed in photodynamic inactivation of microorganisms

71 cases in 13 European countries all in travelers returning fromthe island and none of them lethal [124] Current updates of firstdetection or confirmation of the presence of other disease vectorscan be found at the European Mosquito Bulletin website [125]

In the photosensitized insect eradication PS which are alreadyregistered as food additives such as chlorophylls chlorophyllinsand their copper complexes [126127] or phototherapeutic agentsas hematoporphyrin IX (HpIX) or 5-aminolevulinic acid (5-ALA)used as a precursor of protoporphyrin IX (PPIX) [22] havebeen especially successful They have a low cost productionlack of mutagenic activity high efficiency and high level ofsafety to humans and in general to other mammalian species[22128]

Typically experimental designs of laboratory studies or stud-ies under semi-field conditions use fly larvae or adult mosquitoeslaboratory-reared or collected in breeding sites which are broughtinto contact with solutions containing free-base porphyrins orporphyrin incorporated into baits An initial period of dark incu-bation more or less extended to allow the uptakeingestion of PS isfollowed by irradiation with natural or artificial sunlight and deter-mination of the mortality percentage or the median lethal dose(LD50 ie the dose at which death is produced in 50 of the exper-imental organisms) (Fig 4) Many studies also inspect the amountof porphyrin taken up the anatomical site where it binds and itsclearance

Hematoporphyrin IX (HpIX) has been tested on Ceratitis capi-tata (Mediterranean fruit fly) and Bactrocera oleae (olive fly) [59] Asugarprotein bait containing 8 M HpIX led to 100 mortality of Ccapitata and B oleae flies within the first day after 1 and 2 h of expo-

sure to 2080 E sminus1 mminus2 respectively However a milder irradiancesuch as 760 E sminus1 mminus2 was also found effective in decreasing thenumber of surviving flies when the irradiation time was prolonged

tobiol

ttttaaftpstphedpmdpo

TL


o 2 h The lower photosensitivity of B oleae flies was possibly due tohe smaller amount of ingested HpIX andor to its darker pigmenta-ion HpIX was localized in the cuticle the midgut the Malpighianubes and the adipose tissue of the flies [59] The same PS waslso tested on Stomoxis calcitrans (house fly) [109] Flies fed with

concentration range from 47 to 75 M of HpIX and irradiatedor 1 h at an irradiance of 1220 E sminus1 mminus2 underwent total mor-ality after 2ndash3 days Only at the highest HpIX dose a significantercentage of dead flies was observed for the 1 h of light expo-ure This insect has a more darkly pigmented body and larger sizehan C capitata and B oleae Besides HpIX other meso-substitutedorphyrins with two three and four positive or negative chargesave been used for insect photosensitization [109129] The mostfficient porphyrin was 510-bis(1-methylpyridinium-4-yl)-1520-iphenylporphyin (Di-Py+-Me-Di-Phadj) a dicationic amphiphilicorphyrin which at the concentration of 085 mM caused 100ortality on C capitata within 1 h of irradiation at an irra-

iance of 1220 E sminus1 mminus2 The photoinsecticidal efficiency oforphyrins seems to increase with the increasing hydrophobicityf the molecule either by the reduction in the overall number

able 2ist of porphyrinic derivatives used in non-clinical photodynamic inactivation experimen

Porphyrin


of positive or negative charges or by the replacement of the 1-methylpyridinium moiety with phenyl rings

The factors that appear to affect the efficiency of insectsrsquo pho-tosensitivity by porphyrins have been identified by Ben Amoret al [59109129] serving as a starting point for designing newstrategies for treatment optimization and specificity increase Suchfactors are light irradiance total light dose PS chemical struc-ture (higher photosensitivity associated with higher degree ofhydrophobicity of the porphyrin particularly obvious with theamphiphilic ones) concentration of porphyrin in the bait thick-ness and color of the insect integument and clearance from theorganism in a 24ndash48 h time interval after the porphyrin uptake[130]

HpIX and three more porphyrin derivatives were used in the PDIof different mosquito larvae under field conditions A HpIX LD50 of32 mg Lminus1 was established for the fourth instar of Culex quinque-fasciatus and this porphyrin was the only effective one on A aegypti

larvae [46]

The strong and fast photo-larvicide activity of HpIX againstC capitata immature larval stages has also been reported

ts

Immobilized (references)

No [47]No [505269]

On filter paper [82]On cotton fabric [86108]


Table 2 (Continued)

Porphyrin Immobilized (references)

On polysilsesquioxane plastic films [61]On cotton fabric [87]On azide-modified cellulose nanocrystals [4375]On chloroacetyl cellulose ester chlorides [78]On cellulose laurate esters plastic films [79]

No [71]No [93]No [497193110] and Tri-Py+-Me-PF on silicacoated Fe3O4 nanoparticles [70] and also onCoFe2O4 nanoparticles [111]

No [81]No [105]No [3637717481105] Tetra-Py+-Me wasentrapped into microporous silica gels [88] andinto three alkylene-bridged polysilsesquioxanes[89]


Table 2 (Continued)


On optically transparent indium tin oxideelectrodes [63]

On chitosan membranes [72]Pd(II) TetraTPPCO2H On a polyurethane matrix [83]

No [105]

In hydrophilic polycaprolactone and polyurethane(TecophilicH) nanofibers [60] on electrospunpolymeric nanofiber materials polyurethaneLarithaneTM polystyrene polycaprolactone andpolyamide 6 [76] into three alkylene-bridgedpolysilsesquioxanes [89] and in a silica-gelsupported antimony porphyrin complex SbTPP[9798]

No [445399100102]


Table 2 (Continued)


No [78459959]

No [94] on acid-functionalized multi-walledcarbon nanotubes [96107] on cellulose laurateesters plastic films [77] and on nylon fibers [73]

No [56ndash58101ndash103] and on chitosan [106](E-140 and E-141) On gelatin films and coatings[80]

No [92] on silica- gel and on a Merrifieldresin-based material [90]

[(piHdg

64] The LD50 of HpIX in the food when activated by light47 photons mol sminus1 mminus2) was 0173 mM determined in theeriod entailed from egg hatching to adult ecdysis The correspond-

ng HpIX LD50 during the dispersal period alone was 0536 mMpIX elicited a mortality of 9087 which was mainly concentrateduring prepupal and early pupal stages Loci in the brain and in theut were damaged by ROS [64]

Awad et al [67] demonstrated the efficiency of HpIX and ofa formulation based on HpIX powder sugar and other additivesas larvicidal substances on Culex pipiens Egyptian field strains

exposed to 3 9 and 18 h of natural sunrise A HpIX concentration of10 and 100 M decreased the larval survival by 94 and 993 at theend of 5 days respectively On the other hand concentrations of 1and 10 M of HpIX-formulation caused a decrease in larval survival


F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm

ig 4 Schematic illustration of the in vivo PDI experiments on disease-transmitting i

f 927 and 992 respectively after 5 days It was proposed that synergistic effect occurred due to the incorporation of sugar andther additives to the HpIX which could reflect the suitability ofsing the sugar as HpIX carrier in the commercial formula [67]

El-Tayeb et al [104] studied the effect of HpIX on the fleshy Parasarcophaga argyrostoma in adult stage A concentration of0 mM of HpIX caused a mortality of 83 and 96 of the treatedies after exposure to natural sunlight with an irradiance of 2365nd 1935 W mminus2 respectively Histological studies showed the highbility of HpIX to accumulate in the insect organs and to causeigh extent damage in the alimentary canal tissue The increase

n irradiance of light and in irradiation time enhanced fly mortal-ty So although the most efficient HpIX concentration to control argyrostoma was 10 mM concentrations of 1 and 001 mM wereufficient to control Musca domestica and Culex pipiens respectively68] More recently larvae of the mosquito Aedes caspius have beenfficiently photoinactivated using 1 mM of HpIX-formulation Thearval mortality has improved by increasing light irradiance andxposure times [48]

In order to be applied to endemic areas with scarce eco-omic resources PS must be inexpensive Some researchers havesed natural and modified PS centered on chlorophyll deriva-ives (chlorophyllin and pheophorbide) and sunlight Chaoborus spaphnia sp and Culex sp larvae were photosensitized with chloro-hyllin (15 mg Lminus1) incubated in darkness overnight and were then

rradiated for 3ndash4 h with artificial sunlight (PAR 14966 W mminus2V-A 3267 W mminus2 and UV-B 077 W mminus2) After incubation chloro-hyllin eliminated the different organisms at remarkably lowoncentrations The LD50 value in Culex sp larvae was about

88 mg Lminus1 in Chaoborus sp larvae of about 2418 mg Lminus1 and

n Daphnia 055 mg Lminus1 It was found that during the pupariumosquito larvae are relatively insensitive to chlorophyllin treat-ent and during metamorphosis the chrysalid is encapsulated

ectors contaminated foodstuff and infected fish in aquaculture PS photosensitizer

and totally stops food uptake Probably because of that the accu-mulation of chlorophyllin inside the organism is limited and thephotodynamic effect is reduced As well as in other studies somedark toxicity has also been observed Other small animals likeDaphnia and fish (Chaoborus) larvae are also affected by chloro-phyllin [66] On the other hand unlike fish larvae more maturefish are unharmed and survive chlorophyllin treatment at concen-trations at which for example Culex larvae are severely affectedThis has been shown in field tests in Nigeria in which chloro-phyllin was used on Anopheles larvae and successfully destroyedthe mosquito larvae in a treated pond without harming any of theother aquatic organisms [51] Moreover in addition to the inactiva-tion of mosquito larvae Wohllebe et al [65] demonstrated for thefirst time that the photodynamic treatment of C crystallinus larvaewith 24 mg Lminus1 of chlorophyllin solution gives a LC50 with 026 MJ(PAR + UV-A + UV-B) and induces necrosis and apoptosis in theseorganisms Chlorophyllin was orally taken up and accumulated inthe intestine (a dose of 32 mg Lminus1 chlorophyllin with 3 h irradiationinduced apoptosis in the intestinal cells) [65]

Besides chlorophyllin magnesium chlorophyllin zinc chloro-phyll and copper chlorophyll have been tested on C crystallinuslarvae [23] After 6 h of dark incubation and 3 h of light expo-sure (360 W mminus2) the LD50 value of magnesium chlorophyllin wasabout 2225 mg Lminus1 and for zinc chlorophyll 1753 mg Lminus1 Cop-per chlorophyll (LD50 01 mg Lminus1) was shown to be toxic alsowithout light Chlorophyllin (LD50 1488 mg Lminus1) was lyophilizedimmediately after extraction and its photodynamic efficiencyremained constant over a 30-day period representing an increasein photodynamic efficiency of 50 compared to magnesium chloro-

phyllin isolated with standard methods (no lyophilization) and 18compared to zinc chlorophyllin The results showed that about30 W mminus2 of solar radiation which is lt10 of full sunlight is wassufficient to induce lethal photodynamic effects in larvae (around

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4 E Alves et al Journal of Photochemistry and Pho

0 of the mosquito larvae) Depending on the attenuation in aater body photodynamic action can also take place below theater surface Temperature has also been shown to influence the

ctive chlorophyllin uptake by the larvae [23]As well as HpIX and chlorophyllin the hematoporphyrin deriva-

ive dimethyl ether (HpDE) has also been shown to be a highotential photopesticide against the larvae of the leafminer fly Liri-myza bryoniae [99] The insects exposed to a sugar bait containing5 mM of HpDE and irradiated for 30 min with broad spectrum visi-le light at an irradiance of 30 mW cmminus2 (54 J cmminus2 light dose) died100 mortality) 1 day after irradiation (in the case of the females)nd 4 days after the irradiation (in the case of the males) Thebserved differences in the mortality kinetics between female andale flies were possibly due to the body size and biological activ-

ty of females [99] The treatment efficiency strong also stronglyepended on the type of PS used explaining the different attrac-iveness of the insects for the bait according to the specificity of theS contained on it [99] In line with this need different baits con-aining porphyrin to inactivate disease-transmitting vectors haveecently been designed and tested

Lucantoni et al [47] prepared a formulation constitutedy 5-(1-tetradecylpyridinium-4-yl)-101520-tris(1-methylpyridi-ium-4-yl)porphyrin (Tri-Py+-C14-Py+-Me) and powdered foodellet (PFP) First the LC50 of C14 porphyrin in solution wasested on photosensitized 3rdndashearly 4th instar A aegypti lar-ae LC50 values of 01 M (015 mg Lminus1) and 05 M (077 mg Lminus1)ere obtained after irradiation intervals of 12 and 1 h with

0 mW cmminus2 artificial white light respectively Tri-Py+-C14-Py+-e was shown to be active after ingestion by the larvae and

aused irreversible lethal damage to their intestinal tissuesmidgut and caecal epithelia) The porphyrin carrier formula-ion (25 mg of PFP in 500 mL of 50 M of Tri-Py+-C14-Py+-Me

PF-50-Tri-Py+-C14-Py+-Me) and a 5 M of Tri-Py+-C14-Py+-e solution were both 100 effective up to two weeks and

he amount of Tri-Py+-C14-Py+-Me required to prepare theF-50-Tri-Py+-C14-Py+-Me was 10 times smaller than the Tri-Py+-14-Py+-Me required to treat the incubation medium In the sameirection Fabris et al [50] associated 5-(1-dodecylpyridinium--yl)-101520-tris(1-methylpyridinium-4-yl)porphyrin (Tri-Py+-12-Py+-Me) with two distinct carriers a model of a pharmaceuticalral vehicle (Eudragitreg S100 EU) and a model of foodstuff carriercat food pellet Friskiesreg CF) These phorphyrin-carrier formulates50 M Tri-Py+-C12-Py+-Me dose) were tested against overnighted Anopheles gambiae and Anopheles arabiensis larvae exposed tounlight (30ndash110 mW cmminus2) for 05ndash3 h These conditions led toigh photoinactivation efficiency against laboratory reared A gam-iae and A arabiensis larvae (almost complete mortality) On wildfield-collected) larvae the formulation EU-50-Tri-Py+-C12-Py+-MeU caused 100 mortality on A gambiae M and S forms but CF-50-ri-Py+-C12-Py+-Me showed variable results depending on the sitehere the larvae were collected Not only the association of the PSith suitable carriers promoted a fast and selective internalization

f formulates by the Anopheles larvae which guaranteed their deathpon subsequent sunlight exposure but also the nature of the car-ier affected the overall efficacy of the porphyrin formulates Thisas shown by the administration of a 11 mixture of CF-50-Tri-Py+-

12-Py+-Me and laboratory food for larvae (TetraMinreg) inducing anxtensive mortality of both laboratory reared and wild Anophelesarvae These results indicated the primary importance of palata-ility in the design of oral larvicide formulations [50]

According to Lucantoni et al [47] an insoluble baited insec-icidal formulate should be actively consumed by the larvae an

fficient and cost-effective employment of the PS suitable forpplication in household water storages for drinking and otheromestic purposes unlikely to affect the organoleptic proper-ies of the stored water selected or manipulated on different

ogy C Photochemistry Reviews 22 (2015) 34ndash57

porphyrin carriers in a way to standardize the particle dimensionto a size range that is especially palatable for mosquito larvae (eg5ndash50 m) thus reducing the risks of uptake by non-target organ-isms

22 Water disinfection

Water reuse is increasingly becoming an essential require-ment Not only in undeveloped countries but also in developedcountries treating drinking water and wastewater may be chal-lenging Chlorine-based agents the most commonly used waterdisinfectants are effective against a broad range of microbes areinexpensive and easy to use but lack effectiveness against para-sites and lead to toxic by-products Alternatives to chlorine such asozone chloramines chlorine dioxide and UV irradiation also haveadvantages and drawbacks dealing with cost efficiency stabilityease of application development of microbial resistance and muta-tions triggered by UV irradiation and nature of formed by-products[131132] In this way new technologies for water treatment haveemerged in recent years [133]

The PDI of microorganisms in the context of water treatmentis considered to cause minimal environmental impact and over-come economic ecological and public health issues Contrarily tothe conventional methods no toxic by-products are formed andno microbial resistance is developed The possibility of immobiliz-ing efficiently PS in insoluble supports is also an advantage of PDIBy this way it is possible to remove the PS after treatment fromenvironmental waters and to reuse it which turns this technol-ogy environmentally friendly and cost-effective Besides the use ofsunlight as light source in PDI has to be considered in order to estab-lish a sustainable antimicrobial protocol Since sunlight penetratesdeeply into the water column a nearly uniform illumination canbe achieved and high water volumes may be treated This approachbecomes inexpensive since it is based on the use of a low cost visiblelight source Moreover for PS as porphyrins with the Soret absorp-tion band in the 420ndash430 nm spectral region there is an efficientinteraction with blue light which has a high penetration depth intonatural waters and is the most effective in the visible range in theinactivation of microorganisms by cationic porphyrins [134]

The possibility of treating wastewater for reuse in agri-culture (crop irrigation) was mentioned in the research workcarried out by Alouini and Jemli [3637] Accordingly theauthors demonstrated the efficient PDI of helminth eggsby the tetracationic 5101520-tetrakis(1-methylpyridinium-4-yl)porphyrin (Tetra-Py+-Me) under visible light illumination inclear water and in secondary treated wastewater In addition todifferent concentrations of PS and light intensities the influence ofturbidity (content of suspended solids) agitation (aeration) con-centration of dissolved oxygen and the ultrastructural changesof two types of eggs of parasites was also investigated Suspen-sions of Ascaris lumbricoides and Taenia spp were irradiated withartificial white light (0ndash05 W cmminus2) with Tetra-Py+-Me (5ndash30 M)for 15 min in clear water and 30 min in wastewater There was adestruction of around 28 of Ascaris eggs with 033 W cmminus2 and10 M of Tetra-Py+-Me after 15 min in clear water and after 30 minin wastewater The increase in the light irradiance to 05 W cmminus2

caused 47 of destruction of Ascaris eggs in wastewater after30 min of irradiation This increase turned negligible the differ-ence in the sensitivity of Ascaris eggs in clear and wastewater Anincrease in the oxygenation of the wastewater and in the agita-tion process improved the photosensitivity of Taenia eggs In factthese effects were more evident when the concentration of PS

was also increased The process was optimized under the follow-ing conditions dissolved oxygen was maintained at 7 mg Lminus1 theTetra-Py+-Me concentration at 30 M and the egg suspension wasmixed at 300 rpm The efficiency of the photoprocess was mainly

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ontrolled by the Tetra-Py+-Me concentration the irradiation timend the light intensity Also the dissolved oxygen concentrationater quality and the type of eggs can influence the sensitivity

f helminth eggs to photosensitization Ascaris eggs were foundo be more sensitive to photosensitization than Taenia eggs Theame authors also used Tetra-Py+-Me in several concentration (100 and 100 M) and sunlight irradiation (1235 mW cmminus2) up to40 min to inactivate fecal coliforms and fecal streptococci on sec-ndary wastewater samples After 60 min at 50 and 100 M aecrease of 294 and 24 logs in fecal bacteria counts was observedespectively After 240 min a total cell survival reduction (gt40 lognits) was achieved with both concentrations The 50 M concen-ration was considered to be more suitable to reduce fecal coliformsn wastewater since it allows obtaining a good treatment yield andt is more economic [37] The suspended solids (turbidity) werehe most influential solution parameter on the efficiency of thehotochemical process Turbidity reduces light penetration whicheduces the PS excitation and the absorption by the helminth eggs36] The decrease in log counts of fecal coliforms was asymp10 after 1 hf phototreatment by 5 M Tetra-Py+-Me when suspended solidseached 50 mg Lminus1 [37]

Albeit these results were promising the practical applicationf photodynamic treatment to disinfect microbiologically pollutedaters depends on many factors the removal of the PS after photo-ynamic treatment to avoid the release of PS to the environmenthe use of photostable PS (ie PS which do not bleach under irradi-tion) the impact of this procedure on the structure of the naturalon-pathogenic microbial communities the toxicity of the PS toquatic organisms at doses which induce marked mortality onicrobial pathogens the effect of physical and chemical proper-

ies of environmental waters and the possibility of using sunlights light source [2832]

In a pioneering work Bonnett et al [72] incorporated meso-etraarylporphyrins with amino and hydroxy substituents andlso a tetra sulfonated zinc phthalocyanine (ZnPcS4) into chi-osan membranes for the specific purpose of water disinfection

odel reactors for a large-scale water-flow system were designed static photoreactor system with 7 mL of bacterial suspension3500 cells mLminus1) and a circulating water photoreactor systemith 455 mL of bacterial suspension (105 CFU ndash colony form-

ng units ndash per mL) representing the significant levels of waterontamination After 30 min of irradiation with white lighthe chitosan membrane containing the 5101520-tetrakis(4-minophenyl)porphyrin (TetraTPPNH2) caused a reduction to300 CFU mLminus1 However the membrane prepared with ZnPcS4as much more effective and was able to completely inactivate

he bacteria after 30 min When the more efficient membraneas stored in the dark for nine months the photodynamic actionas still detectable demonstrating its thermodynamic stabilityith that model system the photoinactivation with immobilized

S can be used to lower microbial levels in water flow systems72]

The bactericidal effect of silica gel-supported antimony por-hyrin complex SbTPPSiO2 under visible-light irradiation overegionella pneumophila has been reported by the group of Yasudat al [9798] Laboratory and environmental field experiments in

cooling tower of 800 L capacity were carried out using fluores-ent lamps as light source and also in a public fountain filled with3 m3 of water and sunlight irradiation In vitro the inactivationf L pneumophila with SbTPPp-SiO2 (10 mg) reduced the survivalate to 06 after 60 min of irradiation In the cooling tower after 10ays the concentrations of Legionella were reduced to the detection

imit and these levels were kept until the irradiation had finishedn the public fountain the bacterial concentrations were reduced tohe detection limit 12 days after the SbTPPSiO2 catalyst had beennstalled in the fountain The bacterial concentrations were kept at


lt30 CFU 100 mLminus1 for 3 months until the removal of the catalystfrom the fountain [9798]

Pyrrolidine-fused chlorin derivatives (TPFPC) showed afterimmobilized on either silica gel or Merrifield resin-based materialsignificant activity against Escherichia coli (30 log reductions) with40 mW cmminus2 after 180 min The photostability of the materials andtheir preserved activity after 3 successive cycles was envisaged asa promising option for water disinfection [9092]

Since 2004 our research group has developed a broad-spectrum of PS namely cationic porphyrins which can efficientlyinactivate bacteria [1871] bacterial endospores [135] viruses[1735136] and fungi [105] One of the most effective compoundsa tricationic porphyrin with a pentafluorophenyl group (51015-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrintri-iodide Tri-Py+-Me-PF) has been tested on bacteria and virusesto check the viability recovery and resistance development afterrepeated incomplete photoinactivation cycles The bacteria andviruses that were inactivated to the detection limit with Tri-Py+-Me-PF did not recover viability after one week and the resistanceis not enhanced after 10 sub-lethal photosensitization treatments[3841] At the initial stage of our work porphyrins were used tophotoinactivate fecal coliforms and fecal enterococci in wastewa-ter samples from a secondary-treated sewage plant [93] Two ofthe cationic porphyrins used (Tri-Py+-Me-PF and Tetra-Py+-Me)inactivated 94ndash998 of the fecal coliforms at 5 M upon whitelight at low irradiance (4 mW cmminus2) after 270 min of irradiationdemonstrating high efficiency In this study two rapid monitoringmethods were used to oversee the bacterial photoinactivationgalactosidase activity as an indicator of the presence of fecalcoliforms and leucine incorporation as an indicator of bacterialactivity The advantages of using these methods are their easierand faster performance against the determination of CFU givingan excellent relation with fecal indicators abundance [93] Fromthese first studies the experimental conditions were standard-ized for testing the antimicrobial photoinactivation in vitro ofour porphyrins in further studies cell cultures ranged from 107

to 108 CFU mLminus1 porphyrin concentrations ranged from 05 to50 M white light irradiation had an irradiance of 4 mW cmminus2

and the maximum exposure time was 270 min (648 J cmminus2) Theincubation time of the organisms with the porphyrins was 10 minin the dark previously to irradiation in accordance with theliterature [14] Specific conditions beyond these will be describedon the text

Later seven synthetic cationic meso-substituted porphyrinswith one to four charges were tested on Enterococcus faecalisand E coli (107 CFU mLminus1) The results showed that the tri-(Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me) and the tetra-cationic PS(Tetra-Py+-Me) at 50 M were the most efficient ones reducingE coli survival by 7 log CFU mLminus1 after 90 min with Tri-Py+-Me-PFand Tri-Py+-Me-CO2Me and after 270 min with Tetra-Py+-Me [18]The complete photoinactivation of bacteria with low light irra-diance (4 mW cmminus2) suggests that PDI of fecal bacteria can be apossibility for wastewater disinfection under natural light condi-tions

It was also reported the photoinactivation of a recombinant bio-luminescent E coli strain whose light emission decreased morethan 4 log with the three porphyrins used (Tetra-Py+-Me Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me) These results were achieved bothwith artificial white light (40 mW cmminus2 648 J cmminus2) and with sun-light (asymp62 mW cmminus2 10044 J cmminus2) after 90ndash270 min with 50 Mof PS but Tri-Py+-Me-PF was the most efficient compound [71] Thesame series of cationic porphyrins were also tested on bacterio-phages isolated from wastewater using white light and 50 M of

porphyrin [17] Again the tetra- and tricationic derivatives inac-tivated the T4-like phage to the limits of detection (reduction ofasymp7 log)

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As nanotechnology emerges as an opportunity for waterreatment purposes [131133137138] new materials based on

agnetic nanoparticles with different porphyrins covalentlymmobilized have been recently developed in order to be eas-ly removed from the water matrix for subsequent reuse [1870]hree different hybrids have been prepared and tested in micro-osm conditions being the multi-charged nanomagnet hybridased on Tri-Py+-Me-PF quite effective against Gram-positive andram-negative fecal bacteria (5 log decrease for E faecalis and E colit 20 M of PS) when using white light irradiation (648 J cmminus2)his hybrid also presented antiviral activity inactivating the T4-ike bacteriophage to the detection limit (asymp7 log of inactivation)70]

In order to use this approach in operative field conditionshe multi-charged nanomagnet-Tri-Py+-Me-PF hybrid has beenecently tested [111] Preliminary results showed that this hybrids effective in Allivibrio fischeri photoinactivation in at least a six-ycle reuse causing a cumulative bacterial reduction higher than7 log under low light irradiance (40 mW cmminus2)

Also Tri-Py+-C14-Py+-Me has been encapsulated within silicaicroparticles forming a conjugate with ca 09 m diameter The

onjugate loaded with 12 M C14 added to a water sample contam-nated with methicillin-resistant S aureus (MRSA) (108 cells mLminus1)romoted a tight association of the bacterial cells with the silicaicroparticlendashporphyrin system and further irradiation with vis-

ble light (30 min 100 mW cmminus2) caused a 3 log reduction in theurvival of MRSA cells in the water sample The conjugate showedo be stable in aqueous medium for at least 3 months easily recov-red by filtration of the aqueous suspension and kept the highntibacterial photo-activity when reused [91]

Another example of recovery and reuse of immobilizedorphyrin used protoporphyrin IX (PPIX) attached to acid-unctionalized multi-walled carbon nanotubes (NT-PPIX) as potentntiviral agents to be used on surface disinfection as a coating orn water treatment [96] Influenza virus strain X-31 AAichi268H3N2) was irradiated with visible light and 1 mg mLminus1 of NT-PPIXp to 90 min This treatment caused more than a 250-fold reduc-ion in the effective infectious viral dose after a 30 min exposureo light The percentage of cells that could be infected by 200 ngf virus was only about 1 A pre-exposure of NT-PPIX for 90 minhowed a partial loss of photoactivity but its effect on the virusas still equivalent to a reduction in the infectious viral dose by

lmost 50-fold Reusability studies of NT-PPIX showed to be depen-ent on photobleaching of the porphyrin moiety and on the yield ofecovery of NT-PPIX after each use Besides being effective bacte-icidal agents as previously proved on Staphylococcus aureus [107]hese conjugates may be applied as reusable antivirals in wastew-ter treatment as they can be easily recovered without leaving anyoxic by-products [96]

Although there is a tremendous need for scientific knowledgen disinfection of hospital wastewater there is only one reportrom our group on the use of photodynamic treatment on thisype of effluent The efficiency of photoinactivation on four multi-rug resistant isolated strains of E coli Pseudomonas aeruginosa Sureus and Acinetobacter baumannii was evaluated in buffered solu-ion and in hospital wastewater using 50 M of Tetra-Py+-Me andhite light (648 J cmminus2) [139] The results showed an efficient inac-

ivation of multidrug-resistant (MDR) bacteria in buffered solutionreduction of 6ndash8 log CFU mLminus1) and in the wastewater in whichhe photoinactivation of the four bacteria was also effective andhe decrease in bacterial number occurred even sooner This dis-imilarity was assigned to dissolved compounds in the hospital

astewater such as antibiotics

Aquaculture activity is increasing worldwide motivated by therogressive reduction of natural fish stocks This activity suffersowever substantial financial losses resulting from fish infections


by pathogenic microorganisms The misuse of a wide variety ofantibiotics in aquaculture has raised several problems such as theemergence of MDR bacteria in aquaculture environments the risein antibiotic resistance in fish pathogens the transfer of theseresistance determinants to bacteria of land animals and to humanpathogens and modifications of the bacterial flora both in watercolumn and in sediments [140] Moreover although commercialvaccines against the most common pathogens are available for fishvaccination is not an option for fish larvae one of the fish life stagesmost prone to microbial infection as it is unfeasible to handle largenumbers of these small-sized and frail organisms Furthermore fishlarvae do not have the ability to develop specific immunity [141]Consequently PDI can be an alternative approach to treat fish lar-vae The treatment can be applied as a preventive approach againstbacterial infections during larvae production before releasing themin the aquaculture tanks thereby improving the overall productionof adult fish and the sustainability of fish farming

As a consequence strategies to control fish pathogens areneeded and PDI can be an option to treat diseases and to preventthe development of antibiotic resistance by pathogenic bacteria[142] As the PS could be degraded and used in a low concentra-tion it would be innocuous to animals to fish consumers and tothe environment However the idea is not to use the PS diluted ordispersed in the aquaculture water but instead use it immobilizedin solid supports in order that it could be recovered and reused suc-cessively without being discarded Aquaculture water treated withimmobilized PS under sunlight makes this approach attractive costeffective and eco-friendly

The photodynamic disinfection of water from fish farms aswell as the prevention and treatment of localized fungal infec-tions (saprolegniosis) in fish with porphyrins as photosensitizingagents was presented in 2006 demonstrating the inactivationin vitro of pathogenic fungi and bacteria and the photo-treatmentof spontaneously or artificially infected fish in a pilot aquaculturepond [81] The PS chosen were Tetra-Py+-Me and Tri-Py+-C14-Py+-Me (005ndash10 M) The treatment of infected rainbow trout(Oncorhynchus mykiss) consisted in two protocols preventive andcurative (Fig 4) In the preventive protocol artificially infectedfish in 1000 L capacity tanks were dark incubated for 10 min with02 M of Tri-Py+-C14-Py+-Me and were not irradiated or wereincubated for 10 min with 044 M of Tetra-Py+-Me and irradi-ated for 1 h with white light (50 mW cmminus2) The incubation andirradiation treatment was repeated daily for ten consecutive daysstarting from the first day after the infection In the curative proto-col infected fish were dark incubated with 044 M Tetra-Py+-Mefor 10 min in an 80 L pool irradiated for 1 h and moved to a 1000 Ltank (treatment repeated daily for six consecutive days) or darkincubated with 04 M of Tri-Py+-C14-Py+-Me for 24 h in a 150 Ltank not irradiated and then moved to a 1000 L tank [81]

Experiments with Saprolegnia (asymp108 zoospores mLminus1) indicatedthat Tri-Py+-C14-Py+-Me caused a decrease of the survival of thefungal cells by about 6 logs after phototreatment with 10 M[81] The preventive protocol determined a reduction of theinfected percentage to 10 and 13 respectively after one weekwith irradiated Tetra-Py+-Me and unirradiated Tri-Py+-C14-Py+-Me respectively After two weeks all the infected individuals fromall the experimental groups recovered from the infection Withthe curative protocol a complete remission of the infection wasinduced within one week followed by the complete healing of theulcerated lesion Gradual photobleaching of both porphyrins wasobserved but photodegradation products were not toxic [81]

In the case of treating infected fish by this approach either in

a preventive or curative way the PS was added to the water tankfollowed by irradiation with artificial white light [81] (Fig 4) Inthat case the porphyrins were able to interact with the pathogencells that had colonized the lesion in fish and either cause their

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nactivation or prevent their proliferation The induced-infectionsy Saprolegnia sp in trouts were external on the dorsal region thushe infection was treated by exposing the lesion in the fish to waterdded with porphyrin and after a period of irradiation

For aquaculture water disinfection using PDI the PS may bedded to water in a solid membrane or dispersed in particles toisinfect this matrix before being in contact with fish Microbiolog-

cally contaminated water would be decontaminated prior to beingelivered to the fish tank culture However treating fish diseaseshrough PDI is another concern that has not been object of enoughcientific research

Studies carried out in our laboratory indicated that nine bacte-ial species (Vibrio anguillarum V parahaemolyticus Photobacteriumamselae subsp damselae P damselae subsp piscicida Aeromonasalmonicida E coli Pseudomonas sp S aureus and E faecalis) iso-ated from fish-farming plant water are effectively inactivatedup to 7 log units) with the tricationic porphyrin Tri-Py+-Me-PFt 50 M under a low light irradiance (40 mW cmminus2) [49] Theultivable fraction of the heterotrophic bacteria of the same aqua-ulture plant including pathogenic and non-pathogenic bacteriaas also inactivated by PDI but the efficiency of the inactiva-

ion varied during the sampling period The seasonal variationf photoinactivation efficiency can be due to differences in bac-erial community structure but also due to variations of theater physico-chemical properties The results clearly show that

t is more difficult to photoinactivate the complex natural bac-erial communities of aquaculture waters than pure cultures ofacteria isolated from these waters [49] As PDI is not selec-ive for pathogenic microorganisms the non-pathogenic microbialommunity of aquaculture waters can also be affected As non-athogenic bacteria have an important ecological role in theiogeochemical cycles in aquaculture waters namely in semi-

ntensive systems a careful evaluation of the environmental impactust be conducted before PDI implementation in these systems

reliminary results from our group showed that not all bacterialopulations are affected by PDI In aquaculture water treated withri-Py+-Me-PF and exposed to light a reduction on the numberf bacterial genotypes relatively to the non-treated water samplesas been observed indicating that dominant bacterial populationsere affected by PDI However bacterial population is also affected

y simple light exposure [49]The effect of physico-chemical properties of aquaculture waters

n PDI efficiency has also been evaluated [110] The PDI assays wereesigned having into account the annual variability of pH (65ndash75)emperature (13ndash22 C) salinity (10ndash35 g Lminus1) and oxygen con-entration (2ndash6 mg Lminus1) values in the fish farms where the wateras collected To monitor the PDI kinetics the bioluminescence of fischeri was measured The variations in pH temperature salin-

ty and O2 did not significantly affect the PDI of V fischeri (asymp7 logeduction in all conditions) with 50 M of Tri-Py+-Me-PF underhite light (40 mW cmminus2) The suspended solids in the aquacul-

ure water reduced the efficiency of PDI in relation to clear aqueousolutions (buffered solution) On the other side a good responseas obtained in aquaculture water by increasing the PS concentra-

ion to 20 M The kinetics of the photoinactivation of A fischerin aquaculture water using two different light sources (artificial

hite light and solar light) was also tested The inactivation of fischeri under solar light (40 mW cmminus2) was compared to thatbtained with artificial white light using the same total light dosehe results obtained with 20 M of porphyrin showed that usinghe same total light dose (648 J cmminus2) both light sources inacti-ate A fischeri to the detection limit As it is intended that this

echnology will be used in real aquaculture context using solarrradiation as light source is expected to make this water disinfec-ion approach economically sustainable in terms of energy demand110]


To overcome the enteric septicemia of catfish columnaris dis-ease and the presence of odor-producing cyanobacteria in ponds ofcatfish (Ictalurus punctatus) production the antibacterial and algi-cidal activity of Tri-Py+-C12-Py+-Me has been evaluated on the fishpathogens Edwardsiella ictaluri and Flavobacterium columnare andon S aureus for comparison [69] The same PS was also used on theodor-producing planktonic cyanobacterium Planktothrix perornataand on a representative green alga Selenastrum capricornutum Tri-Py+-C12-Py+-Me was used in concentrations ranging from 001 to1000 M and the samples were irradiated with fluorescent lampsat a photon flux density of 16ndash30 E mminus2 sminus1 Minimal inhibitoryconcentrations were obtained as follows 98 mg Lminus1 for E ictaluri05ndash10 mg Lminus1 for F columnare and 01 mg Lminus1 for S aureus Thegrowth inhibition after 96 h irradiation with 007 and 02 mg Lminus1

of C12 porphyrin inhibited 50 of both P perornata and S capri-cornutum respectively (total inhibition was observed at 10 mg Lminus1

in both cases) While in vivo tests or other toxicity test were notreported the preliminary findings showed the broad spectrumactivity of Tri-Py+-C12-Py+-Me and suggested applications includesanitizing empty tanks before restocking and treatment of otheraquaculture systems dealing with similar disease outbreaks [69]

Tri-Py+-C12-Py+-Me already mentioned as an effective pho-toactivated antimicrobial agent for aquaculture [69] and disease-transmitting insect vectors [50] has also been tested on theprotozoan Ciliophora Colpoda inflata and Tetrahymena thermophileand on the Crustacea Branchiopoda Artemia franciscana and Daph-nia magna which are used in routine toxicity assessment infreshwater ecosystems [52] Following incubation with Tri-Py+-C12-Py+-Me for 60 min (01ndash10 M range) the organisms wereirradiated with visible light (10 mW cmminus2) for 60 min as well Tri-Py+-C12-Py+-Me caused a growth inhibition ge90 against C inflatacysts or trophozoites with 03ndash06 M 3 h after irradiation wasended In turn T thermophila vegetative cells required 3 M for a50 inhibition of growth 46 h after irradiation The complete inacti-vation of D magna was achieved with 06 M of Tri-Py+-C12-Py+-Mewhile in A franciscana photosensitivity was not detected up to10 M Fluorescence microscopy analyses clearly revealed a dam-aged morphology induced on cysts of C inflata by 1 M of PS andon trophozoites of C inflata by 06 M T termophila also revealeddamage with 6 M of Tri-Py+-C12-Py+-Me D magna accumulatedthe PS in the digestive tract and exoskeleton and A fransciscanarevealed accumulation especially on the exoskeleton The resis-tance of A franciscana to the phototreatment was explained by thevery low amount of the ingested PS along with its ability to adapt toextreme conditions These findings emphasized the need for care-fully tailored irradiation protocols taking into account the natureof the specific water basin particularly in what refers to its bioticcharacteristics [52]

Recently chlorophyllin-mediated PDI has been suggestedas a new promising treatment to control ichthyophthiriosis awhite spot-causing disease in many freshwater fish species by theprotozoan parasite Ichthyophthirius mulftifiliis Different life stagesof the parasite were tested trophonts (encysted stage in the host)and tomites (motile infective stage) Samples were incubated60 min in the dark with 05ndash10 g mLminus1 followed by irradiationwith simulated sunlight (PAR 14966 W mminus2 UV-A 3267 W mminus2and UV-B 077 W mminus2) for 30 min The photodynamic effect on thecells was evaluated by fluorescence microscope after staining withthe fluorescent dye acridine orange Transmitted light microscopyshowed that chlorophyllin completely filled the trophont anddestroyed the nucleus and the cell wall The LC50 value calculatedfor trophonts of I multifiliis was 067 g mLminus1 with a maximum

mortality of about 10 observed in dark conditions Even at achlorophyllin concentration of 2 g mLminus1 in the medium 100of the tomites were dead The results of the in vitro experimentsshowed that the photodynamically active chlorophyllin reduces

4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p


he number of parasites already at very low concentrations For therst time it was demonstrated that the photodynamic treatmentf I multifiliis with chlorophyllin is very effective against tomontsnd tomites of these fish parasite [95]

3 Elimination of food-borne pathogens

Conventional thermal methodologies used for food preservationre effective against most of the food-borne pathogens Howeverajor drawbacks deal with uncontrolled chemical reactions within

he food matrix which affects their nutritional value New technolo-ies for food safety such as ultraviolet irradiation ionizing radiationr high pressure are only at research stage most of them fail to inac-ivate bacterial spores viruses and biofilms and also require highnvestment and specialized equipment [143]

In this sense PDI emerges as a new tool for food decontam-nation in a non-thermal way [143] PDI of microorganisms foroodstuff decontamination contrarily to the conventional ther-

al methodologies satisfy the increasing consumer demand foricrobiologically safe fresh-like foods with minimal modification

f nutritional and organoleptic properties [144] Moreover whenompared with other non-thermal new technologies for food safetyuch as high pressure PDI is effective to inactivate bacterial sporesiruses microbial biofilms and antimicrobial resistant microorgan-sms which are the most common causal agents in bacterial foodoisoning outbreaks

PDI of common food pathogens (yeasts molds and bacteria)as been reported in vitro and in vivo on foodstuff (sprouts fruitsegetables meat products) by directly applying the PS or immo-ilizing it on edible solid supports (Fig 4) and also on the surfacef packaging material In the last 10 years representative naturalype porphyrins such as HpDEe PPIX sodium chlorophyllin (NaChl)r 5-ALA as prophyrin precursor and also synthetic analogs haveeen used in scientific research related with microbiological foodontrol

HpDE (25ndash71 M) has been tested on several strains oficromycetes with distinct morphological type Aspergillus flavus

richothecium roseum Fusarium avenaceum Rhizopus oryzae [7]lternaria alternata and Acremonium strictum [45] using visi-le light (30 mW cmminus2) and 15 min of irradiation (27 J cmminus2) on06 spores mLminus1 suspensions incubated for 20 min to 18 h before

rradiation After 18 h incubation with HpDE dark toxicity inhibitedhe conidia germination of the different microfungi Concentrationsf 51 M caused 100 inhibition of spore germination after irradi-tion of Aspergillus [7] R oryzae and A alternata [45] Dark toxicityercentages of asymp15 and 20 were observed at this concentrationor both fungi respectively [45] The germination of microfungi Fvenaceum and T roseum spores were inhibited by 71 M up to0ndash100 (with 30 of dark toxicity) [7] For A strictum only theighest concentration (108 M) induced a photoinhibition of 90ith 9 of dark toxicity [45] This dark toxicity was envisaged as a

pore germination inhibitor which could prevent germination untilhey have been washed or diluted out of the surface [7]

High inhibition of conidia germination was also observed afterDI when using PPIX (1ndash4 M) on Fusarium poae and Fusar-

um culmorum isolated from infected barley seeds The sporesasymp105ndash106 mLminus1) incubated 30 min were irradiated with visibleight at 150 W mminus2 (50ndash200 kJ mminus2) At a PS concentration of 1 Mnd an illumination dose of 200 kJ mminus2 germination decreasedo 40 for F poae and 65 for F culmorum At 4 M 96 and5 of conidia were eliminated for F poae and F culmorumespectively Also at micromolar concentrations PPIX sensitizes

he photo-oxidation of proteins and lipids in hydrated coni-ia when illuminating the spore suspension with light doses of0ndash200 kJ mminus2 disturbing the permeability of membranes and sup-ressing spore germination [94]


5-ALA has been stated to improve the nutritive value of food andconsidered as a possible tool to decontaminate wheat seeds priorto their use into preparation of sprouts with minimal damage towheat germination and quality Wheat (Triticum aestivum L varZentos) seeds significantly contaminated with several microfungiwere soaked and incubated with 6 mM 5-ALA for 4 h After this timethe viability of Mucor spp Penicillium spp Aspergillus spp Rhizo-pus spp Acremonium spp Fusarium spp Alternaria spp Myceliasterilia and other fungi significantly decreased asymp10 log CFU gminus1 ofseeds The sensitivity of fungi to 5-ALA treatment varied consider-ably among fungi Besides antifungal activity 5-ALA demonstratedto be a growth stimulator of wheat seedlings and roots withoutimpairment of the vigor of germination and the viability of seedsIt also caused an increase of the chlorophyll content favoring therate of photosynthesis and of the activity of antioxidant enzymeswhich can promote cellular detoxification from ROS [44] The selec-tion of this particular pro-drug took into account the fact that PS caninteract with the food matrix Being colorless and odorless 5-ALAwould not change the organoleptic properties if sprayed on food[53] Moreover its water solubility stable shelf-life non-bleachingcharacter simplicity of production and the possibility of being aconstituent or food additive make 5-ALA a potential candidatefor the control of food pathogens by photosensitization withoutdamage to food constituents and unwanted coloration [145]

The efficiency of 5-ALA has been tested in vitro againstSalmonella enterica Bacillus cereus vegetative cells and spores Lis-teria monocytogenes cells and biofilms and on packaging material(adhered cellsspores and biofilms) The approach of adherence topackaging consisted on soaking or submerging trays of polyolefin (amixture of polyethylene and polypropylene) in 107ndash108 CFU mLminus1

cells or spores solutions for 30 min followed by dark incuba-tion up to 60 min drying 20 min and irradiation with visible light(20 mW cmminus2) up to 20 min In vitro results showed that B cereuscells were inactivated by asymp6 logs with 3 mM incubated 60 min andirradiated 20 min and spores asymp3 logs with 75 mM after 30 minincubation and 15 min irradiation Cells adhered to packagingmaterial were inactivated by 4 log after 10 min incubation with3 mM porphyrin and 15 min irradiation Increasing the concentra-tion to 75 mM and the incubation time to 30 min caused 27 logdecrease on spores adhered to packaging [55] An efficient inacti-vation (6 log) of Salmonella enterica serovar Typhimurium resistantto tetracycline (107 CFU mLminus1) was achieved after 60 min incuba-tion with 75 mM of 5-ALA and 20 min irradiation (24 J cmminus2) [54]In the case of L monocytogenes in vitro tests showed that cellswere inactivated by 4 logs with 75 mM 5-ALA incubated 120 minand irradiated 20 min Cells adhered to packaging material wereinactivated by 37 logs after 15 min incubation with 10 mM PSand 15 min irradiation (18 J cmminus2) With the same concentrationa 31 log decrease in viable cells was observed in biofilms formedin 48 h incubated 30 min in the dark and irradiated for 15 minApproximately 59 log CFU cmminus2 of biofilm-associated cells wereadhered onto a plastic coupon The different effectiveness betweenphotosensitization of biofilms and the cells adhered to the surfaceof packaging material was explained by the polysaccharide matrixof biofilms acting as a diffusion barrier for PS and reducing its accu-mulation inside bacteria [53] The fluorescence intensity analysisof endogenous PS in these three microorganisms revealed that Bcereus produces endogenous porphyrins 10 times more efficientlythan L monocytogenes or S enterica [54100] explaining its highersusceptibility to 5-ALA-based photosensitization compared to theother two bacteria

The photodynamic effect of chlorophyllins applied for inactivat-

ing food-borne pathogens was firstly reported by Loacutepez-Carballoet al [80] In a way to improve food preservation the authors devel-oped and applied new antimicrobial PS-containing edible filmsand coatings based on gelatin incorporating sodium magnesium

tobiol

cCwEsoAoisaEpEagcapcr1f1fPTipmbc

pfocl(pf(vlnsussiLarliogd

aSb(epb


hlorophyllin (E-140) and sodium copper chlorophyllin (E-141)ell suspensions of S aureus and L monocytogenes (108 CFU mLminus1)ere spread on the surface of nutritive agar plates E-140- and

-141-containing gelatin films (80 g mLminus1) were placed on theurface of the inoculated nutritive agar plates and irradiated for 5r 15 min at several light intensities 10000 30000 and 50000 lxdditionally the inhibitory effect of 80 g mLminus1 E-140-gelatin filmsn early-stationary phase cells of S aureus and L monocytogenesnoculated on slices of cooked frankfurters was tested Frankfurterlices were inoculated with bacterial samples (50 L 106 cells) andfter inoculum penetration the sample surfaces were wrapped in-140 edible films and exposed to 30000 lx for 15 min Chloro-hyllin E-140 exerted a greater inhibiting effect than chlorophyllin-141 on the bacterial viability Differences in the antibacterialctivity of the two chlorophyllins could be due to E-140 having areater ability than E-141 to generate 1O2 under the experimentalonditions tested Water soluble E-140 and E-141 reduced S aureusnd L monocytogenes viability by 5 log and 4 log respectively Bothrocedures of wrapping or coating the food sample with E-140-ontaining gelatin films and coatings were successful although theesults of the antimicrobial studies were slightly different The E-40 gelatin film was placed on the surface of a previously inoculatedrankfurter sample the combined treatment of wrapping with E-40 gelatin film and irradiation produced an approximately 15 logall in the viability of S aureus and L monocytogenes None of theS treatments showed effect on the viability of E coli or Salmonellahe antimicrobial films with potential application as wraps or cast-ngs to avoid contamination on the surface of fresh and processedroducts would increase their shelf-life and safety Additionallyinimal impact on the visual sensory properties of the food has

een observed with the low concentrations used of incorporatedhlorophyllin [80]

Besides immobilized chlorophyllin derivatives sodium chloro-hyllin (NaChl) has also been tested in vivo on the surface ofruits and vegetables [58] The inactivation of B cereus and aer-bic mesophils on nectarines iceberg lettuce cherry tomatoesauliflowers and cherries and the impact of the treatment on shelf-ife (ie disease-free period) of the product were evaluated B cereusasymp107 CFU mLminus1) were inoculated on the surface of these foodroducts and they were soaked in the NaChl solution (015 mM)or 5 min dried for 20 min and irradiated with the visible light20 mW cmminus2) for 5 min A 168ndash288 log reduction on bacterial sur-ival was observed depending on the surface structure and theight-surface interaction specificity A reduction of 148ndash247 log ofaturally distributed aerobic mesophils was also achieved Also thehelf-life of all treated products could be extended in some casesp to 70 compared to non-treated ones [58] In another studytrawberries were inoculated with L monocytogenes for 30 minoaked in 1 mM NaChl for 5 min and irradiated for 20 min with vis-ble light (12 mW cmminus2 144 J cmminus2) Photosensitization decreasedisteria by 98 (18 log) Naturally occurring yeasts microfungind mesophils were inhibited by 86 (086 logs) and 97 (17 log)espectively This photo-treatment extended the strawberry shelf-ife by 2 days The total antioxidant activity of treated strawberriesncreased 19 No impact on the amount of phenols anthocyaninsr surface color was detected The re-growth rate of yeast and fun-al survivors slightly decreased Furthermore photosensitizationid not affect strawberry color or taste [103]

NaChl has also been tested in vitro and on surfaces of pack-ging material with effective results on B cereus Listeria andalmonella enterica The experimental approach used in the studieselow was the same already described for 5-ALA [53ndash55] NaChl

0075ndash75 M) was tested against B cereus (107 CFU mLminus1) veg-tative cells and spores in vitro or attached to the surface ofackaging material incubated 2ndash60 min and irradiated with visi-le light (20 mW cmminus2) B cereus vegetative cells and spores were


inactivated in vitro by 7 log with 075 M after 2 and 5 min irra-diation respectively after 2 min incubation in both cases The4-log photoinactivation of B cereus cells on the surface of pack-aging materials required a concentration of NaChl 10-fold higher(75 M) PDI with 75 M of NaChl 5 min of dark incubation and5 min irradiation caused 5 log reductions in the concentration ofattached endospores Endospores are more resistant than vege-tative cells to photoinactivation since a higher concentration ofNa-Chl is required than for vegetative cells for the same level ofinactivation to be attained [56] Bacillus spores were susceptible to5-ALA-based PDI as well [55] The comparison of 5-ALA and Na-Chl based photoinactivation of B cereus indicates some advantagesof the later PS this means that using NaChl can shorten the darkincubation time from 20 to 2 min reduce the irradiation time from20 to 5 min and lower the optimal concentration (0075 M versus75 mM for 5-ALA) [145]

Listeria biofilms were totally removed from the surface of pack-aging material at higher NaChl concentrations and after longerincubation times L monocytogenes cells were inactivated by 7 logusing 075 M of PS and 30 min irradiation for a thermo-resistantstrain and 5 min for a thermo-sensitive one Decontamination ofpackaging material from thermo-resistant L monocytogenes cellsadhered to the surface reveals that a NaChl concentration of015 M is necessary to reduce the 4 log of bacteria with 15 minirradiation while the same reduction against the thermo-sensitivestrain could be achieved within 5 min With this irradiation timeListeria biofilms were also susceptible to photosensitization andsurfaces could be totally cleaned from them when 150 M of NaChlis used (45 logs) [101] Additionally a thermo-resistant strain ofBacillus was less susceptible to PDI than the thermo-resistant Lis-teria strain [57]

A comparison between the antimicrobial efficiency of PDIwith conventional surface cleaning products indicates that NaChl-based photosensitization is much more effective against B cereusattached to the surface of materials than washing with water orusing 200 ppm of sodium hypochlorite [56] In the case of Listeriastrains (thermo-resistant or thermo-sensitive) washing with waterdiminishes pathogens by lt1 log 200 ppm of sodium hypochloriteby 17 log and NaChl-based photosensitization by 45 log [101]

Chitosan as a natural compound may replace chemicalpreservatives and may be used to obtain eco-friendly productsbut it should not interact with food ingredients or alter foodorganoleptic features [7] A chlorophyllinndashchitosan complex (with0001 NaChlndash01 chitosan NaChlndashCHS) has been tested againstSalmonella enterica (asymp107 CFU mLminus1) upon visible light irradiation(96 mW cmminus2) for 30 min [106] The photoinactivation treatment(15 min of incubation with NaChl followed by irradiation) led to a105 log reduction of S enterica The dark toxicity of the NaChlndashCHScomplex reduced cell viability by also 105 log An extension of theincubation time to 120 min favored the inactivation of Salmonellato 139 log An extremely high antibacterial efficiency was demon-strated after photoactivation with the NaChlndashCHS complex (73 logreduction of microbial population) after 120 min irradiation A pho-toactivated NaChlndashCHS complex in a slightly acidic environmentwas suggested as a useful tool against S enterica The synergeticantibacterial effect of CHS and NaChl supposedly relies on the com-plexation of these two compounds governed by thermodynamicequilibrium and results in a uniform distribution of the short-chaincomponent (NaChl) among the chains of the oppositely chargedlong-chain component (CHS) present in high excess [106]

More recently the photodynamic antimicrobial efficiencyamong 5-ALA NaChl and a combined treatment of 5-ALA plus NaChl

has been tested against L monocytogenes and S enterica Cells wereincubated with NaChl (75ndash0075 M) or 5-ALA (75 mM) and fur-ther irradiated with visible light (20 mW cmminus2) Salmonella wasmore sensitive to 5-ALA (asymp66 log viability decrease with 75 mM

5 tobiology C Photochemistry Reviews 22 (2015) 34ndash57

47N(Bb7

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bullbullbull

bullbull

bull

3s

snrrsaparwafcm8esactra

gmoc

Table 3Potential non-clinical applications of porphyrin-based photoactivecompoundsmaterials

References

Water and environmentAlgicides [69]Control of fish infections in

aquaculture systems[81]

Control of insect pests aroundpoultry-producing facilities in barnsfeedlots manure storage areasgreenhouses

[99]

Disinfection of polluted waters fromfish-farming ponds

[4981110]

Filters for water treatment [85]Larvicidesinsecticidespesticides [2346ndash48505964ndash6899104109]Wastewater treatment [3637709396]Water disinfection [52637296]Food safetyDecontamination of food-related

surfaces (packaging material)[55ndash577580101]

Decontamination of ready-to-eatfruits frozen fruits pastry productsor similar

[58103]

Decontamination of sprouts [4494]Wraps or castings for fresh and

processed products[80]

Domestic industrial and healthcare settingsSelf-cleaning surfaces [436375ndash77]More specificallyHygiene products or packaging

materials[82]

Polymeric materials (syringes IV bagsand tubing catheters hemodialysisfilters and gloves)

[617379]

Privacy curtains in hospital rooms [73]Protective clothes and masks [73]Sources of nosocomial infections

textile (bedding surgeons gownspillows)

[7379]

Walls floors instruments [88]


0 min irradiation) than to NaChl (asymp22 log viability decrease with5 M upon 40 min irradiation) Listeria was more sensitive toaChl (7 log drop with 075 M 5 min irradiation) than to 5-ALA

asymp38 log with 075 M 20 min irradiation and 60 min incubation)oth bacteria were inactivated to an undetectable level when com-ined treatment was applied (incubation time of 60 min with5 mM of 5-ALA and 015 M of NaChl 40 min of irradiation) [102]

Besides HpDE PPIX 5-ALA and NaChl novel cationic por-hyrins were synthesized to be used on food contaminantss such the biological evaluation of five cationic porphyrinsas tested on Penicillium chrysogenum conidia Two dif-

erent series of cationic porphyrin groups were preparedrom 5101520-tetrakis(4-pyridyl)porphyrin and 5101520-etrakis(pentafluorophenyl)porphyrin A 50 M of PS was usednder white light at an irradiance of 200 mW cmminus2 over 20 minhowing that the most effective PS caused a 41 log (Tetra-Py+-Me)nd a 34 log (Tetra-Py+-C5) reduction in the concentration ofiable conidia An increase of the porphyrin carbon chain led to aeduction of the inactivation of conidia in both PS series [105]

From these reports one can detach the factors that influence thefficiency of PDI of food-borne pathogens

irradiation time (and consequently light dose) [53ndash555880]the surface specificity of food as shown by in vivo studies [58]cell-bound PS concentration as shown in bacteria [58] and in fungi[45]the use of powerful light sources (light emitting diodes) [103]the size of the N-alkyl chain on the porphyrin structure as shownon the photoinactivation of fungi spores mainly by affecting theirbinding to cellular material [105]initial concentration in the case of 5-ALA [55] and incubationtime (or concentration of endogenously produced porphyrins)[53ndash55]

Applications for domestic industrial and healthcareettings

At a time when environmental protection energy efficiencyustainability and entrepreneurship are guiding principles ofew technological solutions it makes sense that the collabo-ation between researchers and businessmenentrepreneurs iseinforced combining innovation and creativity In this partner-hip which is an increasingly evident reality the design productionnd commercialization of multi-functional reusable and durableroducts arise as emerging opportunities Portuguese companiess Success Gadget and Revigreacutes are examples of this close collabo-ation The first of these companies developed silica nanoparticlesith different active ingredients (anti-mosquito antimicrobial

ntifungal) with applications in the textile industry (clothing andootwear) construction (paints) and health (antifungal socks antihilblain gloves and socks for diabetic foot) The innovation of theseaterials is linked to wash off resistance (they withstand up to

0 washes) and when applied in paints varnishes or mortar theirffect lasts up to five years [146] The second company Revigreacutesells self-cleaning tiles suitable for outdoor use (fac ade claddingnd external cladding) since its titanium oxide coating when inontact with sunlight promotes the degradation of gaseous pollu-ants Also whenever the tile is wet a wet film formed allows theemoval of dirt These properties reduce the maintenance costs andlso promote cleaning in areas of difficult access [147]

Despite these innovative examples there is still a long way to

o with regard to fast effective and long-lasting elimination oficroorganisms on surfaces whether in the household industrial

r healthcare settings The application of the photodynamic prin-iple to this context seems to be a promising option according to

OtherHand hygiene [74]

studies conducted in recent years Table 3 gathers the potentialnon-clinical applications of porphyrin-based photoactive com-poundsmaterials suggested by the referred literature

31 Porphyrin-embedded fabric

The thought of incorporating porphyrins into different textilematerials to create protective clothing in the household indus-trial and hospital settings including masks gowns caps beddingand other textiles was suggested nearly 10 years ago The firstantimicrobial porphyrin textiles were reported by Bozja et al [73]named as light-activated antimicrobial materials Novel materialswere synthesized by the grafting of PPIX and zinc protoporphyrinIX (Zn(II)-PPIX) to nylon fibers and their antimicrobial effective-ness tested against S aureus and E coli Samples of the textileswere inoculated with bacteria and irradiated with incandescentlight (at 10000 40000 and 60000 lx for 30 min or at 40000 lxfor 5 15 and 30 min) At 40000 lx these fibers showed increasedantimicrobial activity against S aureus with increasing exposuretime The samples grafted with PPIX-ethylene diamine (PPIX-ED)were able to inactivate more than 95 of S aureus after 30 minof irradiation upon 60000 lx but no reduction on E coli survivalwas observed The samples grafted with the zinc(II) complex ofPPIX-ethylene diamine were able to eliminate 94 of S aureus at

40000 lx but a light intensity of 60000 lx was required to attain areduction of 30 on E coli survival The zinc samples were moreeffective than the corresponding free-base With suitable changesthese materials have been suggested to be used for privacy curtains

tobiol

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srCbobwCtsw((arfag(aasad2coe9ta

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n


n hospital rooms laboratory coats for medical personnel or otherber or plastic surfaces within the hospital environment consider-

ng that the normal roomsrsquo illumination is approximately 30000 lx73]

Other authors relied on the possibility of creating pioneeringelf-sterilizing fabrics from cotton fiber due to its resistance toepeated washing or exposure to cleaning products [8687108]otton fabrics which essentially consist of cellulose fibers cane easily modified by substitution of a relative small numberf their numerous ndashOH nucleophilic groups Meso-arylporphyrinsearing acetylenic units (ZnTri-Py+-MeCCH and Tri-Tolyl-OCCH)ere grafted on cotton fabric with azide groups via ldquoClick-hemistryrdquo Covalently attached porphyrin accounted for 70 ofhe total amount of PS and the remainder (around 30) corre-ponded to porphyrins only sequestered in the cellulose fibereb This material was impregnated with E coli and S aureus

asymp106 CFU per 10 cm Petri dish) and irradiated with white light1000 lx) for 24 h For both bacteria a ca 074 log reduction waschieved after irradiation [87] Soon afterwards the same groupeported a modification in porphyrin immobilization envisagingurther industrial applications In this way covalently neutralnionic and cationic 135-triazinylporphyrin derivatives wererafted on non-modified cotton fabrics using cyanuric chloride246-trichloro-135-triazine) as linking agent The photodynamicctivity of these new cotton fabrics was tested in vitro against Sureus with white light for 24 h (016 mW cmminus2) Sterile photo-ensitive textiles (35 times 35 cm) previously autoclaved for 15 mint 120 C impregnated with bacterial inoculum (asymp106 CFU mLminus1)eposited in a sterile Petri dish were then incubated at 37 C for4 h under white light irradiation in wet atmosphere All surfacesaused a photo-bactericidal effect at a quantity as low as asymp10 molf active compound per fabric square sample After 24 h of lightxposure percentages of bacterial survival reduction were 3737 and 100 for anionic neutral and cationic surfaces respec-ively The three materials did not show any photodynamic activitygainst Gram-negative bacteria [86108]

Mosinger et al [84] produced a novel polyurethane nanofab-ic using the NanospiderTM electrospinning industrial technology148] which is used for the preparation of fibers with diametersanging from nanometers to a few microns and for materials with

high surface area and porous structure [149] The material wasoped with 5101520-tetraphenylporphyrin (TPP) The nanofab-ics composed of polymeric nanofibers had 003 mm thickness2 g mminus2) and contained 012 TPP A hydrophilic variant was alsoroduced containing in addition 06 sodium dodecyl sulfate andhe photodynamic function of this nanofabric variant was testedgainst E coli Small pieces of nanofabrics were inoculated withacteria and illuminated with white light for 60 min After irra-iation and incubation on agar plates no bacterial growth wasbserved on the nanofabric pieces Daylight was at least as effec-ive as artificial light TPP incorporated in both nanofabrics variantsere able to produce 1O2 with high efficiency Among the excep-

ional properties of these nanofabrics are the large specific surfacerea light weight chemical specificities low cost and mechanicalexibility The same authors studied three polyurethane nanofab-ics containing respectively 01 zinc(II) phthalocyanine (ZnPc)1 ZnTPP and a mixture of PS (01 ZnPc and 01 ZnTPP)nother polyurethane nanofabrics was also prepared but contain-

ng two independent layers one doped with 01 ZnPc and thether doped with 01 ZnTPP All nanofabrics were placed on agarlates and inoculated with a suspension of E coli in an undeter-ined concentration and illuminated with white light for 30 min

ll PS-doped nanofabrics were able to kill bacteria on their surfaceshen exposed to white light [85]

Later Jesenskaacute et al [76] prepared electrospin polymericanofibers of polyurethane LarithaneTM (PUR) polystyrene (POS)


polycaprolactone (PCL) and polyamide 6 (PA6) doped with 1 TPPThe PUR POS and PCL nanofiber materials were able to inhibitcompletely the E coli growth after 60ndash90 min of irradiation withwhite light while lower antibacterial activity was found for PA6nanofiber materials The photodynamic effect was attributed to 1O2production and all tested nanofiber materials exhibit prolongedantibacterial properties after being treated with long-durationirradiation due to the formation of H2O2 even in the dark Thephotoinactivation efficiency also depended on oxygen permeabil-ity and diffusion coefficients as on the diameter of the polymericnanofibers Soon after the same group [60] selected anothermedical-grade nanofiber material polyurethane Tecophilicreg andalso polycaprolactone (PCL) both loaded with TPP to study thephotoinactivation of non-enveloped DNA viruses (mouse poly-omaviruses 105 plaque forming units PFU) and enveloped DNAviruses (baculoviruses pVLVP1 104 PFU) Viral suspensions wereapplied to the surface of small pieces of the nanofiber textiles dopedwith 1 TPP and were irradiated with white light for 30 min Thevirus were retrieved from the surface of the textiles and used toinfect 3T6 fibroblasts (for the mouse polyomavirus) and the Sf9insect cell line (for baculovirus) [60] Singlet oxygen released fromthe surface of doped polyurethane Tecophilicreg and PCL upon irradi-ation efficiently inactivated both the non-enveloped and envelopedviruses and both types of polymer nanofibers are nontoxic toviruses in the absence of light The results showed that baculovirusis more resistant to photoinactivation than the polyomavirus Thetextiles were also tested after a year of storage at room temperaturein the dark with the same results which indicate their long-termphotovirucidal efficiency [60]

32 Porphyrin-embedded paper

Another possible application of microbial PDI is in hygieneproducts or packaging materials Cellulose is thus envisaged as anexceptional sustainable material as it is degradable and renew-able [82] Paper or cardboard can thus be sanitized prior touse to avoid contamination In this scope a tricationic por-phyrin 5-(4-aminophenyl)-101520-tris(1-methylpyridinium-4-yl)porphyrin (Tri-Py+-Me-NH2) was grafted on cellulose filter paperby using cyanuric chloride without previous chemical modificationof the cellulosic support as already mentioned [108] Sterile pho-tosensitizing filter paper disks (05 cm diameter) impregnated withE coli and with S aureus (105 CFU mLminus1) were deposited on sterilePetri dishes and then incubated at 37 C for 24 h under white lightirradiation (95 J cmminus2) in wet atmosphere A strong photobacteri-cidal effect was observed after light exposure since no survivingbacteria were detected on grafted filter paper [82]

33 Porphyrins immobilized in other support materials

Krouit et al reported several works considering the preparationof plastic materials based on natural and synthetic porphyrins ableto inactivate Gram-positive and Gram-negative bacteria [77ndash79]Photobactericidal surfaces were prepared containing differentamounts of PPIX units (019ndash11) covalently linked to cellu-lose esterified with lauric acid [77] Besides plastic materialincorporating synthetic cationic porphyrin derivatives (014ndash057Tri-Tolyl-Py) was also prepared in order to achieve a better pho-tobactericidal activity In this work the cellulose was derivatizedwith chloroacetyl groups [78] Based on the first work the groupprepared cellulose laurate esters plastic films grafted by differ-ent carbon-spacer arms (4- or 11-carbon) to synthetic porphyrins

(008ndash058) [79] The photodynamic activity of these polymerswas tested against E coli and S aureus Disks with 2 cm diame-ter were cut out of the porphyrinated plastic films and depositedonto nutrient agar seeded with the target strain and irradiated with

5 tobiol

vmdwtfpl[t

nbcrttfaaatwcSsfcsSasf

wtbariM(dbobPiflnbtidewi

tae(itpi


isible light (17 mW cmminus2) for 24 h at 37 C The growth of theicroorganisms was examined visually under the porphyrinated

isks The cationic porphyrinic chloroacetyl cellulose chloridesith porphyrin content higher than 020 inactivated the two bac-

erial strains whereas films with lower porphyrin content wereound more active against S aureus than against E coli [78] In com-arison the films based on PPIX lauryl cellulose esters needed at

east a porphyrin content value of 052 to photoinactivate E coli77] These results confirmed the higher photobactericidal effect ofhe cationic porphyrinic plastic films [79]

The possibility of incorporating PS-modified-celluloseanocrystals into paper fabrics and plastics was highlightedy Feese et al [75] who created a conjugate cellulose nanocrystals-ationic porphyrin (CNC-Por) with the aim to rapidly inactivate aange of bacteria with minimal cost As stated cellulose nanocrys-als have several advantages over amorphous cellulose esterhey have a particular degree of molecular control for preciseunctionalization due to their rigidity defined surface structurend dimensions they are readily biodegradable they represent

renewable resource which eases the development of newdvanced materials with photobactericidal activity [75] To testhe performance of these materials a zinc(II) complex of a cationicater-soluble ethynylphenylporphyrin (ZnTri-Py+-Me-CCH) was

ovalently attached to azide-modified cellulose nanocrystalsaline buffered suspensions of E coli S aureus and Mycobacteriummegmatis (asymp108 CFU mLminus1) were incubated in the dark for dif-erent time intervals with 20 M CNC-Por which refers to theoncentration of the porphyrin on CNC-Por present in the suspen-ion Then they were illuminated with white light (60 mW cmminus2)ix log units reduction in viable cells were observed against Sureus (5 min incubation 30 min irradiation) 35 log units for Mmegmatis (45 min incubation 30 min irradiation) and 2 log unitsor E coli (60 min incubation 30 min irradiation) [75]

The PDI of bacteria mediated by these same conjugates (20 M)as further tested as a function of bacterial strain incubation

ime and illumination time on A baumannii multidrug resistant Aaumannii (MDRAB) methicillin-resistant S aureus (MRSA) and Peruginosa (asymp108 CFU mLminus1) with visible light (65 mW cmminus2) [43] Aeduction of 6 log units in viable cells was observed for MRSA (5 minncubation) 5ndash6 log units for A baumannii (30 min incubation) and

DRAB (15 min incubation) and 25 log units for P aeruginosa60 min incubation) at a total light dose of 118 J cmminus2 (30 min irra-iation) Confocal laser scanning fluorescence microscopy of Aaumannii and S aureus incubated with CNC-Por suggested a lackf internalization of the PS supporting the hypothesis that directinding and uptake of the PS are not necessary requirements forDI by CNC-Por On the other hand the ZnTri-Py+-Me-CCH whenn solution and not bound to the cellulose nanocrystals led to theuorescence being localized on A baumannii Even though it wasot determined whether the cationic PS was internalized by theacterium or simply bound to the bacterial cell membrane Sohe production of 1O2 and other radical species in close proxim-ty to the bacteria may be sufficient to result in cell inactivation oreath Despite forming an insoluble suspension CNC-Por conjugatexhibited excellent antimicrobial activity for all strains examinedith the exception of P aeruginosa which was only moderately

nactivated [43]The efficient photodynamic effect of other photoac-

ive surfaces was demonstrated by Funes et al [63] Theuthors used two polymeric porphyrinated films formed bylectrochemical polymerization of free-base 5101520-tetrakis(4-NN-diphenyl)aminophenyl)porphyrin (Tetra-NN-DiPh-film) andts complex with Pd(II) (Pd(II)Tetra-NN-DiPh-film) on optically

ransparent indium tin oxide (ITO) electrodes on microbial cell sus-ensions The polymeric surfaces (07 times 30 times 21 cm2) were placed

nside of E coli and Candida albicans suspensions (asymp106 CFU mLminus1)


and irradiated with visible light (90 mW cmminus2) for different timeperiods The viability of microbial cells irradiated with visiblelight depended on the light exposure Both Tetra-NN-DiPh- andPd(II)Tetra-NN-DiPh-films exhibited a photosensitizing activitycausing a asymp3 log decrease of E coli survival after 30 min of irradi-ation and a asymp2 log decrease in C albicans viability The advantagesof these electrodes are that they represent an appropriated surfaceto obtain mechanically stable electropolymeric films they areoptically transparent to visible light and also that they can beeasily and quickly removed from the media after cell inactivationavoiding permanent photodynamic effects [63]

In another approach to photoinactivate C albicans [61]novel photoactive flexible bridged polysilsesquioxane thin plasticfilms (SSO-PS) doped with 5-(4-carboxyphenyl)-101520-tris(4-methylphenyl)porphyrin (Tri-Tolyl-CO2H) were evaluated inaqueous suspensions (with 5 M of Tri-Tolyl-CO2H) and on twoSSO-PS films (with 26 times 10minus4 (ww) and 52 times 10minus4 (ww) concen-tration of Tri-Tolyl-CO2H in each film) The yeasts treated with5 M of Tri-Tolyl-CO2H were incubated 30 min in dark at 37 Clater irradiated with visible light (90 mW cmminus2) In the case of poly-meric films their surfaces (07 times 25 = 175 cm2) were inoculatedwith yeast suspension (asymp106 CFU mLminus1) irradiated and then placedin saline buffer for serial diluting plating and colony countingWith 5 M of Tri-Tolyl-CO2H (non-cationic porphyrin) an expectedsmall photoinactivation effect (05 log decrease) was observed after60 min The viability of C albicans cells in irradiated SSO-PS filmsdepended on the light exposure although no differences wereobserved between the two SSO-PS films They caused a photosen-sitizing activity of asymp25 log decrease of C albicans survival after60 min of irradiation (324 J cmminus2) [61] Comparatively to the resultsobtained by Funes et al [63] the main difference of the presentSSO-PS films is their versatility as plastic flexible materials at roomtemperature easy to obtain and ability to shape the surfaces Theyalso keep a higher antifungal activity against C albicans

Microporous silica gels prepared by solndashgel process fromtetrakis(2-hydroxyethoxy)silane (THEOS) have been used to immo-bilize the cationic Tetra-Py+-Me [88] THEOS was used to ensuretoughness and greater stability of silica-Tetra-Py+-Me compositesThe antimicrobial activity of these composites was compared withtheir tetramethoxysilane (TMOS) analogs against E coli Nutrientagar inoculated with E coli (106 CFU mLminus1) was poured over thecomposites at the bottom of plastic plates and illuminated with vis-ible light (10000 lx for 0 15 and 3 h corresponding to a light doseof 0 79 and 158 J cmminus2 respectively) Growth of E coli in nutrientagar decreased with increasing light energy for all the compositesOn the dense THEOS composites the 158 J cmminus2 light energy led tothe total growth inhibition (6 log) In the case of TMOS the higherbactericidal effect was established for the weak composites (6 logreduction with the composite with PEG) In general THEOS com-posites with the lower specific surface areas were more effectivethan TMOS analogs The antimicrobial activity of these compos-ites was mainly influenced by total Tetra-Py+-Me content in thebulk specific surface areas and by the thicknesses of the compos-ites Among THEOS composites their flexibility brought about byPEG 600 improved oxygen diffusion All the composites showedgood adhesion to glass and THEOS ones have not shrunk at leastfor 3 months These properties along with a high shape stability ofTHEOS-Tetra-Py+-Me composites make them favorable photosen-sitive materials [88]

The same microbiological method was applied to eval-uate the antibacterial photodynamic effect of mesoporousorganosilicandashporphyrin composites obtained by entrapment ofTPP and Tetra-Py+-Me into three polysilsesquioxanes prepared by

the solndashgel method from 12-bis(triethoxysilyl)ethane (BTE) 16-bis(triethoxysilyl)hexane (BTH) and 18-bis(triethoxysilyl)octaneThe Si-matrices of the porphyrin composites under study are

tobiol

bpan5rcwwtteTptpt[

c((asadPl

tpicicdl1IPC1awscaaa

stcwslppbatp

trt


iocompatible and unresponsive to photosensitization The pre-olymer-porphyrin mixtures were polymerized in a glass Petri dishnd were inoculated with E coli (105 CFU mLminus1) immobilized inutrient agar Irradiation was performed with visible light (with500 lx for 0 15 and 3 h corresponding to 0 43 and 87 J cmminus2espectively) The fluorescence and antibacterial effects of the TPPomposites increased with increasing incident light energy andere dependent on the mesoporosity of the matrix The compositesith the higher mesoporosity are preferable since the matrix con-

rols both oxygen diffusion through the material and quenching ofhe excited PS For that reason TPPndashBTH polymer exerted the high-st biocidal effect (asymp5 log decrease with 87 J cmminus2) and the otherPP composites reduced cell viability to lt1 log Tetra-Py+-MendashBTEolymer was less effective The complete inactivation of the bac-eria attained with TPPndashBTH demonstrates that the photodynamicrocess takes place in the whole volume of the agar layer (1000 mhick) and not only on its contact surface with the TPPndashBTH film89]

In vitro preliminary antimicrobial tests with palladium(II)omplex of 5101520-tetrakis(4-carboxyphenyl)porphyrinPd(II)TetraTPPCO2H)ndashZn2AlPU (polyurethane) composite films04 wt of Pd(II)TetraTPPCO2H for 1 of Zn2Al filler loading) wereble to totally inhibit two strains of S aureus and P aeruginosaeeded on nutrient agar (asymp105 CFU mLminus1) and incubated 24 ht 37 C under continuous white light illumination at an irra-iance of 100 W cmminus2 Considering the low amount of PS thed(II)TetraTPPCO2HndashZn2AlPU film proved to be a highly efficientight activated antimicrobial surface [83]

According to Eichner et al [74] antimicrobial photodynamicreatment might become acceptable as a tool for hand hygienerocedures and also in other skin areas as washing of the hands

s of little use if for example a hospital environment is heavilyontaminated [150] So the possibility of rapidly and efficientlynactivating MRSA enterohemorrhagic E coli (EHEC) and C albi-ans using the photodynamic approach was revealed in vitro withifferent concentrations of Tetra-Py+-Me illuminated with visible

ight (50 mW cmminus2) for 10 s and 60 s A light dose of 05 J cmminus2 and M of Tetra-Py+-Me achieved a PDI of ge3 log of MRSA and EHECncubation with higher concentrations (up to 100 M) of Tetra-y+-Me caused bacterial inactivation by more than 5 log Efficient albicans inactivation (ge5 log) was achieved at a light dose of2 J cmminus2 Since Tetra-Py+-Me has no clinical approval to be used as

medical drug it was suggested that similar porphyrin moleculesith similar properties that can be approved for use in humans

hould be developed in the near future This rapid inactivation pro-edure may be useful for clinical on industrial purposes but it islso feasible in environmental technology Processes with shortpplication times simplicity efficacy and low costs are desirablend needed [74]

In addition to testing the PDI of microorganisms most of thesetudies tried to obtain a mechanistic explanation of the action ofhe PS immobilized on these supports mainly based on the con-ept of lsquophotobactericidal surfacersquo introduced by Midden and Wangho demonstrated that a light-induced flow of 1O2 on the material

urface was responsible for cell inactivation [151] The scientificiterature has disclosed distinctive forms of interaction betweenorphyrins and bacterial cells If on one hand it has been argued thatorphyrins can penetrate the cell wall to the periplasmic space andind to the cytoplasmic membrane [152ndash154] on the other it haslso been demonstrated that they can have a dual location (boundo the cell wall and to the nucleic acids) [155] or do not have toenetrate [156157] or even come into contact with the cells [158]

When porphyrin molecules are immobilized in solid supportsheir availability to bind and eventually penetrate into cells iseduced Thus to produce the same inactivation results the concen-ration used in the supports must be higher than that which would


be used in solution [75] This fact supports the idea that direct bind-ing and uptake of the PS are not necessary requirements for PDI [75]This was justified by the production of ROS on the surface of thematerial mainly 1O2 during activation of the light-porphyrinatedmaterials followed by their dissemination and possible interactionwith the target cell [7778] To interact against the cells and leadto cell death these species would have to be longer-lived andorcould be generated in close proximity to the bacteria [437583]

4 Conclusions

Although not yet established as an antimicrobial therapeu-tic procedure the photodynamic inactivation of microbes andother organisms within the environmental context continues beingimproved and increasingly optimized to meet the urgent needfor alternative options to commonly used antimicrobials to avoiddevelopment of microbial resistance

Among the main advantages of this approach are its nature ofmulti-target cellular damage and the possible use of a powerful andfree light source as sunlight In the 1990s when it was found that aphotosensitizer with positive charges on its structure allowed theefficient inactivation of cells with complex structural features suchas Gram-negative bacteria the research in photodynamic inactiva-tion took a new breath

In addition to being remarkably effective against a multitude ofmicro(organisms) the use of photosensitizers such as porphyrinderivatives allows for the designing of an immense diversity ofstructurally different molecules that can be made more efficientand in the non-clinical framework also to be harmless to theenvironment and to humans and animals if the photosensitizermolecules are immobilized on inert solid supports

Likewise the use of porphyrin concentrations in the micromolarrange or from natural sources enables photodynamic inactivationto be used exclusively for its intended purpose without furtherimpact

The diversity of possible applications that have been proposedin recent years as well as the progress made in this researcharea demonstrate that PDI is a promising method of disinfec-tionsterilization with promising practical application in the shortterm

Acknowledgements

The authors are thankful to the University of AveiroFundac atildeo para a Ciecircncia e a Tecnologia (FCT Portugal) Euro-pean Union QREN COMPETE and FEDER for funding the Centrefor Environmental and Marine Studies (CESAM) unit (projectPest-CMARLA00172013) and the QOPNA research unit (projectPEst-CQUIUI00622013 FCOMP-01-0124-FEDER-037296) ElianaAlves (SFRHBD418062007) is grateful to FCT for her doctoralgrant

References

[1] TJ Dougherty CJ Gomer BW Henderson G Jori D Kessel M Korbelik JMoan Q Peng Photodynamic therapy J Natl Cancer Inst 90 (1998) 889httpdxdoiorg101093jnci9012889

[2] MR Hamblin T Hasan Photodynamic therapy a new antimicrobialapproach to infectious disease Photochem Photobiol Sci 3 (2004) 436httpdxdoiorg101039b311900a

[3] CS Foote Definition of type I and type II photosensitized oxi-dation Photochem Photobiol 54 (1991) 659 httpdxdoiorg101111j1751-10971991tb02071x

[4] AW Girotti Photosensitized oxidation of membrane lipids reaction path-

ways cytotoxic effects and cytoprotective mechanisms J Photochem Pho-tobiol B 63 (2001) 103 httpdxdoiorg101016S1011-1344(01)00207-X

[5] M Perlin JCH Mao ER Otis NL Shipkowitz RG Duff Photodynamic inac-tivation of influenza and herpes viruses by hematoporphyrin Antivir Res 7(1987) 43 httpdxdoiorg1010160166-3542(87)90038-6

5 tobiol


[6] Y Nitzan HM Wexler SM Finegold Inactivation of anaerobic bacteria byvarious photosensitized porphyrins or by hemin Curr Microbiol 29 (1994)125 httpdxdoiorg101007BF01570752

[7] Z Luksiene D Peciulyte A Lugauskas Inactivation of fungi in vitro by pho-tosensitization preliminary results Ann Agric Environ Med 11 (2004)215

[8] Z Luksiene D Peciulyte A Lugauskas Photodynamic inactivation of harmfuland pathogenic microorganisms Vet Med Zootechnol 26 (2004) 58

[9] AC Giese Protozoa in photobiological research Physiol Zool 26 (1953) 1[10] FC Goble JL Boyd Action of certain tetrapyrrole derivatives in experi-

mental Trypanosoma congolense infections Exp Biol Med 100 (1959) 745httpdxdoiorg10318100379727-100-24765

[11] J Tosk A Sherif R Hall B Lau Phototoxicity of hematoporphyrin derivativein larvae of Culex quinquefasciatus Proc Pap Annu Conf Calif Mosq ControlAssoc 54 (1986) 70

[12] M Merchat G Bertolini P Giacomini A Villanueva G Jori Meso-substituted cationic porphyrins as efficient photosensitizers of Gram-positiveand Gram-negative bacteria J Photochem Photobiol B 32 (1996) 153httpdxdoiorg1010161011-1344(95)07147-4

[13] A Minnock DI Vernon J Schofield J Griffiths J Howard ParishSB Brown Photoinactivation of bacteria Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both Gram-negative andGram-positive bacteria J Photochem Photobiol B 32 (1996) 159httpdxdoiorg1010161011-1344(95)07148-2

[14] G Jori C Fabris M Soncin S Ferro O Coppellotti D Dei L Fantetti G ChitiG Roncucci Photodynamic therapy in the treatment of microbial infectionsbasic principles and perspective applications Lasers Surg Med 38 (2006) 468httpdxdoiorg101002lsm20361

[15] GB Kharkwal SK Sharma Y-Y Huang T Dai MR Hamblin Photodynamictherapy for infections clinical applications Lasers Surg Med 43 (2011) 755httpdxdoiorg101002lsm21080

[16] JM Bliss CE Bigelow TH Foster CG Haidaris Susceptibility of Candidaspecies to photodynamic effects of photofrin Antimicrob Agents Chemother48 (2004) 2000 httpdxdoiorg101128AAC4862000-20062004

[17] L Costa E Alves CMB Carvalho JPC Tomeacute MAF Faustino MGPMSNeves AC Tomeacute JAS Cavaleiro Acirc Cunha A Almeida Sewage bacterio-phage photoinactivation by cationic porphyrins a study of charge effectPhotochem Photobiol Sci 7 (2008) 415 httpdxdoiorg101039b712749a

[18] A Almeida JAS Cavaleiro J Rocha CMB Carvalho LAS Costa E Alves ACunha JPC Tomeacute MAF Faustino MGPMS Neves AC Tomeacute Z Lin JPJRainho inventors Nanomagnet-porphyrin hybrid materials synthesis andwater disinfection application Portuguese Patent PT103828 (2009)

[19] T Maisch S Hackbarth J Regensburger A Felgentraumlger W Baumlumler MLandthaler B Roumlder Photodynamic inactivation of multi-resistant bac-teria (PIB) ndash a new approach to treat superficial infections in the 21stcentury J Dtsch Dermatol Ges 9 (2011) 360 httpdxdoiorg101111j1610-0387201007577x

[20] TG St Denis T Dai L Izikson C Astrakas RR Anderson MR HamblinGP Tegos All you need is light antimicrobial photoinactivation as an evolv-ing and emerging discovery strategy against infectious disease Virulence 2(2011) 509 httpdxdoiorg104161viru2617889

[21] M Wainwright Photodynamic antimicrobial chemotherapy (PACT) JAntimicrob Chemother 42 (1998) 13 httpdxdoiorg101093jac42113

[22] A Ormond H Freeman Dye sensitizers for photodynamic therapy Materials(Basel) 6 (2013) 817 httpdxdoiorg103390ma6030817

[23] G Erzinger S Wohllebe F Vollrath S Souza P Richter M Lebert D-P HaumlderOptimizing conditions for the use of chlorophyll derivatives for photodynamiccontrol of parasites in aquatic ecosystems Parasitol Res 109 (2011) 781httpdxdoiorg101007s00436-011-2322-7

[24] LR Milgrom MJ Warren The Colours of Life An Introduction to the Chem-istry of Porphyrins and Related Compounds Oxford University Press IncOxford 1997 httpdxdoiorg101002adma19970091516

[25] RR Allison VS Bagnato CH Sibata Future of oncologic photodynamic ther-apy Future Oncol 6 (2010) 929 httpdxdoiorg102217fon1051

[26] R Darlenski JW Fluhr Photodynamic therapy in dermatol-ogy past present and future J Biomed Opt 18 (2012) 061208httpdxdoiorg1011171jbo186061208

[27] SK Sharma P Mroz T Dai YY Huang TG St Denis MR Hamblin Photo-dynamic therapy for cancer and for infections what is the difference Isr JChem 52 (2012) 691 httpdxdoiorg101002ijch201100062

[28] A Almeida Acirc Cunha NC Gomes E Alves L Costa MA Faustino Phagetherapy and photodynamic therapy low environmental impact approachesto inactivate microorganisms in fish farming plants Mar Drugs 7 (2009) 268httpdxdoiorg103390md7030268

[29] C Carvalho J Tomeacute M Faustino M Neves A Tomeacute J Cavaleiro LCosta E Alves A Oliveira Acirc Cunha A Almeida Antimicrobial pho-todynamic activity of porphyrin derivatives potential application onmedical and water disinfection J Porphyr Phthalocya 13 (2009) 574httpdxdoiorg101142S1088424609000528

[30] O Coppellotti C Fabris M Soncin M Magaraggia M Camerin G Jori LGuidolin Porphyrin photosensitised processes in the prevention and treat-

ment of water- and vector-borne diseases Curr Med Chem 19 (2012) 808httpdxdoiorg102174092986712799034905

[31] G Jori M Magaraggia C Fabris M Soncin M Camerin L Tallan-dini O Coppellotti L Guidolin Photodynamic inactivation of microbialpathogens disinfection of water and prevention of water-borne diseases


J Environ Pathol Toxicol Oncol 30 (2011) 261 httpdxdoiorg101615JEnvironPatholToxicolOncolv30i390

[32] M Magaraggia O Coppellotti C Fabris L Guidolin G Jori Inactiva-tion of microbial pathogens by photosensitized processes environmentalapplications in M Hamblin G Jori (Eds) Photodynamic Inactiva-tion of Microbial Pathogens Medical and Environmental ApplicationsRoyal Society of Chemistry Cambridge 2011 p 403 httpdxdoiorg1010399781849733083-00403

[33] National Office of Forests Bull Tech No (34) 1997 pp 167[34] TS Fies M Mathers The Basics of Efficient Lighting ndash A Reference Manual

for Training in Efficient Lighting Principles National Framework for EnergyEfficiency (Australia) Australian and New Zealand State and Territory Gov-ernments 2009 pp 152

[35] L Costa CMB Carvalho MAF Faustino MGPMS Neves JPC Tome ACTome JAS Cavaleiro A Cunha A Almeida Sewage bacteriophage inac-tivation by cationic porphyrins influence of light parameters PhotochemPhotobiol Sci 9 (2010) 1126 httpdxdoiorg101039c0pp00051e

[36] Z Alouini M Jemli Destruction of helminth eggs by photosensi-tized porphyrin J Environ Monitor 3 (2001) 548 httpdxdoiorg101039B103471P

[37] M Jemli Z Alouini S Sabbahi M Gueddari Destruction of fecal bacteriain wastewater by three photosensitizers J Environ Monitor 4 (2002) 511httpdxdoiorg101039B204637

[38] L Costa JPC Tomeacute MGPMS Neves AC Tomeacute JAS Cavaleiro MAFFaustino Acirc Cunha NCM Gomes A Almeida Evaluation of resistance devel-opment and viability recovery by a non-enveloped virus after repeatedcycles of aPDT Antivir Res 91 (2011) 278 httpdxdoiorg101016jantiviral201106007

[39] FM Lauro P Pretto L Covolo G Jori G Bertoloni Photoinactiva-tion of bacterial strains involved in periodontal diseases sensitized byporphycenendashpolylysine conjugates Photochem Photobiol Sci 1 (2002) 468httpdxdoiorg101039B200977C

[40] LA Pedigo AJ Gibbs RJ Scott CN Street Absence of bacterial resistancefollowing repeat exposure to photodynamic therapy Proc SPIE 7380 (2009)73803H httpdxdoiorg10111712822834 [Photodynamic Therapy Backto the Future]

[41] A Tavares CMB Carvalho MA Faustino MGPMS Neves JPC TomeacuteAC Tomeacute JAS Cavaleiro Acirc Cunha NCM Gomes E Alves A AlmeidaAntimicrobial photodynamic therapy study of bacterial recovery viabilityand potential development of resistance after treatment Mar Drugs 8 (2010)91 httpdxdoiorg103390md8010091

[42] S Lacombe T Pigot New materials for sensitized photo-oxygenation inA Albini (Ed) Photochemistry Specialist Periodical Reports RSC Publish-ing Cambridge 2010 p 307 httpdxdoiorg1010399781849730860-00307

[43] BL Carpenter E Feese H Sadeghifar DS Argyropoulos RA GhiladiPorphyrin-cellulose nanocrystals a photobactericidal material that exhibitsbroad spectrum antimicrobial activity Photochem Photobiol 88 (2012) 527httpdxdoiorg101111j1751-1097201201117x

[44] Z Luksiene H Danilcenko Z Taraseviciene Z Anusevicius A MarozieneH Nivinskas New approach to the fungal decontamination of wheat usedfor wheat sprouts effects of aminolevulinic acid Int J Food Microbiol 116(2007) 153 httpdxdoiorg101016jijfoodmicro200612040

[45] Z Luksiene D Peciulyte S Jurkoniene R Puras Inactivation of possible fun-gal food contaminants by photosensitization Food Technol Biotechnol 43(2005) 1

[46] B Dondji S Duchon A Diabate JP Herve V Corbel JM Hougard R San-tus J Schrevel Assessment of laboratory and field assays of sunlight-inducedkilling of mosquito larvae by photosensitizers J Med Entomol 42 (2005) 652httpdxdoiorg1016030022-2585(2005)042[0652AOLAFA]20CO2

[47] L Lucantoni M Magaraggia G Lupidi RK Ouedraogo O Coppellotti FEsposito C Fabris G Jori A Habluetzel Novel meso-substituted cationicporphyrin molecule for photo-mediated larval control of the dengue vectorAedes aegypt PLoS Negl Trop Dis 5 (2011) e1434 httpdxdoiorg101371journalpntd0001434

[48] TA El-Tayeb NMA El-Aziz HH Awad A study on the dynamics of Aedescaspius larval uptake and release of novel haematoporphyrin Afr Entomol21 (2013) 15 httpdxdoiorg1040010030210108

[49] C Arrojado C Pereira JPC Tomeacute MAF Faustino MGPMS Neves ACTomeacute JAS Cavaleiro Acirc Cunha R Calado NCM Gomes A Almeida Appli-cability of photodynamic antimicrobial chemotherapy as an alternative toinactivate fish pathogenic bacteria in aquaculture systems Photochem Pho-tobiol Sci 10 (2011) 1691 httpdxdoiorg101039c1pp05129f

[50] C Fabris RK Oueacutedraogo O Coppellotti RK Dabireacute A Diabateacute P Di Mar-tino L Guidolin G Jori L Lucantoni G Lupidi V Martena SP Sawadogo MSoncin A Habluetzel Efficacy of sunlight-activatable porphyrin formulateson larvae of Anopheles gambiae M and S molecular forms and An arabiensis apotential novel biolarvicide for integrated malaria vector control Acta Trop123 (2012) 239 httpdxdoiorg101016jactatropica201205011

[51] L Mukwaya Photo-Biological Control of Mosquito Larvae Uganda VirusResearch Institute Entebbe Uganda Available from httpwwwinradcorp

comphotosletterpdf

[52] C Fabris M Soncin G Jori A Habluetzel L Lucantoni S SawadogoL Guidolin O Coppellotti Effects of a new photoactivatable cationicporphyrin on ciliated protozoa and branchiopod crustaceans poten-tial components of freshwater ecosystems polluted by pathogenic

tobiol


agents and their vectors Photochem Photobiol Sci 11 (2012) 294httpdxdoiorg101039c1pp05154 g

[53] I Buchovec E Paskeviciute Z Luksiene Photodynamic inactivation of foodpathogen Listeria monocytogenes Food Technol Biotechnol 48 (2010) 207httpdxdoiorg101111j1751-1097201201154x

[54] I Buchovec Z Vaitonis Z Luksiene Novel approach to controlSalmonella enterica by modern biophotonic technology photosen-sitization J Appl Microbiol 106 (2009) 748 httpdxdoiorg101111j1365-2672200803993x

[55] Z Luksiene I Buchovec E Paskeviciute Inactivation of food pathogen Bacil-lus cereus by photosensitization in vitro and on the surface of packagingmaterial J Appl Microbiol 107 (2009) 2037 httpdxdoiorg101111j1365-2672200904383x

[56] Z Luksiene I Buchovec E Paskeviciute Inactivation of Bacillus cereusby Na-chlorophyllin-based photosensitization on the surface of pack-aging J Appl Microbiol 109 (2010) 1540 httpdxdoiorg101111j1365-2672201004780x

[57] Z Luksiene E Paskeviciute Microbial control of food-related surfacesNa-chlorophyllin-based photosensitization J Photochem Photobiol B 105(2011) 69 httpdxdoiorg101016jjphotobiol201106011

[58] E Paskeviciute Z Luksiene Photosensitization as a novel approach to decon-taminate fruits and vegetables Casopis za procesnu tehniku i energetiku upoljoprivrediPTEP 13 (2009) 50

[59] T Ben Amor M Tronchin L Bortolotto R Verdiglione G Jori Por-phyrins and related compounds as photoactivatable insecticides I Phototoxicactivity of hematoporphyrin toward Ceratitis capitata and Bactroceraoleae Photochem Photobiol 67 (1998) 206 httpdxdoiorg101111j1751-10971998tb05188x

[60] Y Lhotaacutekovaacute L Pliacutestil A Moraacutevkovaacute P Kubaacutet K Lang J Forstovaacute J MosingerVirucidal nanofiber textiles based on photosensitized production of sin-glet oxygen PLOS ONE 7 (2012) e49226 httpdxdoiorg101371journalpone0049226

[61] MG Alvarez ML Goacutemez SJ Mora ME Milanesio EN DurantiniPhotodynamic inactivation of Candida albicans using bridged polysilsesquiox-ane films doped with porphyrin Bioorg Med Chem 20 (2012) 4032httpdxdoiorg101016jbmc201205012

[62] S Eick G Markauskaite S Nietzsche O Laugisch GE Salvi A SculeanEffect of photoactivated disinfection with a light-emitting diode onbacterial species and biofilms associated with periodontitis and peri-implantitis Photodiagn Photodyn Ther 10 (2013) 156 httpdxdoiorg101016jpdpdt201212001

[63] MaD Funes DA Caminos MG Alvarez F Fungo LA Otero EN DurantiniPhotodynamic properties and photoantimicrobial action of electrochemicallygenerated porphyrin polymeric films Environ Sci Technol 43 (2009) 902httpdxdoiorg101021es802450b

[64] LM Pujol-Lereis A Massaldi A Rabossi LA Quesada-Allueacute Photosen-sitizing effect of hematoporphyrin IX on immature stages of Ceratitiscapitata (Diptera Tephritidae) Photochem Photobiol 86 (2010) 639httpdxdoiorg101111j1751-1097200900704x

[65] S Wohllebe C Ulbrich D Grimm J Pietsch G Erzinger R Richter M LebertPR Richter D-P Haumlder Photodynamic treatment of Chaoborus crystallinuslarvae with chlorophyllin induces necrosis and apoptosis Photochem Pho-tobiol 87 (2011) 1113 httpdxdoiorg101111j1751-1097201100958x

[66] S Wohllebe R Richter P Richter DP Haumlder Photodynamic control ofhuman pathogenic parasites in aquatic ecosystems using chlorophyllin andpheophorbid as photodynamic substances Parasitol Res 104 (2009) 593httpdxdoiorg101007s00436-008-1235-6

[67] HH Awad TA El-Tayeb NM Abd El-Aziz MH Abdelkader A semi-fieldstudy on the effect of novel hematoporphyrin formula on the control of Culexpipiens larvae J Agric Soc Sci 4 (2008) 85

[68] TA El-Tayeb The Use of Photoactivable Photopesticides for the Control ofPotentially Noxious Insects (MSc thesis) National Institute of Laser EnhancedSciences Cairo University Cairo 1999

[69] KK Schrader JC Bommer G Jori In vitro evaluation of the antimi-crobial agent AquaFrin as a bactericide and selective algicide for usein channel catfish aquaculture N Am J Aquacult 72 (2010) 304httpdxdoiorg101577a09-0761

[70] CMB Carvalho E Alves L Costa JPC Tomeacute MAF FaustinoMGPMS Neves AC Tomeacute JAS Cavaleiro A Almeida Acirc Cunha ZLin J Rocha Functional cationic nanomagnet-porphyrin hybrids forthe photoinactivation of microorganisms ACS Nano 4 (2010) 7133httpdxdoiorg101021nn1026092

[71] E Alves CMB Carvalho JPC Tomeacute MAF Faustino MGPMS Neves ACTomeacute JAS Cavaleiro A Cunha S Mendo A Almeida Photodynamic inacti-vation of recombinant bioluminescent Escherichia coli by cationic porphyrinsunder artificial and solar irradiation J Ind Microbiol Biotechnol 35 (2008)1447 httpdxdoiorg101007s10295-008-0446-2

[72] R Bonnett MA Krysteva IG Lalov SV Artarsky Water disinfection usingphotosensitizers immobilized on chitosan Water Res 40 (2006) 1269httpdxdoiorg101016jwatres200601014

[73] J Bozja J Sherrill S Michielsen I Stojiljkovic Porphyrin-based light-

activated antimicrobial materials J Polym Sci A Polym Chem 41 (2003)2297 httpdxdoiorg101002pola10773

[74] A Eichner FP Gonzales A Felgentrager J Regensburger T HolzmannW Schneider-Brachert W Baumler T Maisch Dirty hands photody-namic killing of human pathogens like EHEC MRSA and Candida within


seconds Photochem Photobiol Sci 12 (2013) 135 httpdxdoiorg101039c2pp25164 g

[75] E Feese H Sadeghifar HS Gracz DS Argyropoulos RA GhiladiPhotobactericidal porphyrin-cellulose nanocrystals synthesis characteri-zation and antimicrobial properties Biomacromolecules 12 (2011) 3528httpdxdoiorg101021bm200718s

[76] S Jesenskaacute L Pliacutestil P Kubaacutet K Lang L Brozovaacute S Popelka L SzatmaacuteryJ Mosinger Antibacterial nanofiber materials activated by light J BiomedMater Res A 99A (2011) 676 httpdxdoiorg101002jbma33218

[77] M Krouit R Granet P Branland B Verneuil P Krausz New photoantimi-crobial films composed of porphyrinated lipophilic cellulose esters BioorgMed Chem Lett 16 (2006) 1651 httpdxdoiorg101016jbmcl200512008

[78] M Krouit R Granet P Krausz Photobactericidal plastic films based on cellu-lose esterified by chloroacetate and a cationic porphyrin Bioorg Med Chem16 (2008) 10091 httpdxdoiorg101016jbmc200810010

[79] M Krouit R Granet P Krausz Photobactericidal films from porphyrinsgrafted to alkylated cellulose ndash synthesis and bactericidal properties EurPolym J 45 (2009) 1250 httpdxdoiorg101016jeurpolymj200811036

[80] G Loacutepez-Carballo P Hernaacutendez-Munoz R Gavara MJ Ocio Photoacti-vated chlorophyllin-based gelatin films and coatings to prevent microbialcontamination of food products Int J Food Microbiol 126 (2008) 65httpdxdoiorg101016jijfoodmicro200805002

[81] M Magaraggia F Faccenda A Gandolfi G Jori Treatment of microbiolog-ically polluted aquaculture waters by a novel photochemical technique ofpotentially low environmental impact J Environ Monitor 8 (2006) 923httpdxdoiorg101039B606975D

[82] J-P Mbakidi K Herke S Alvegraves V Chaleix R Granet P Krausz SLeroy-Lhez T-S Ouk V Sol Synthesis and photobiocidal propertiesof cationic porphyrin-grafted paper Carbohydr Polym 91 (2013) 333httpdxdoiorg101016jcarbpol201208013

[83] M Merchaacuten TS Ouk P Kubaacutet K Lang C Coelho V Verney SCommereuc F Leroux V Sol C Taviot-Gueacuteho Photostability and photo-bactericidal properties of porphyrin-layered double hydroxidendashpolyurethanecomposite films J Mater Chem B Mater Biol Med 1 (2013) 2139httpdxdoiorg101039C3TB20070A

[84] J Mosinger O Jirsak P Kubat K Lang B Mosinger Bactericidal nanofabricsbased on photoproduction of singlet oxygen J Mater Chem 17 (2007) 164httpdxdoiorg101039b614617a

[85] J Mosinger K Lang P Kubaacutet J Sykora M Hof L Pliacutestil B Mosinger JrPhotofunctional polyurethane nanofabrics doped by zinc tetraphenylpor-phyrin and zinc phthalocyanine photosensitizers J Fluoresc 19 (2009) 705httpdxdoiorg101007s10895-009-0464-0

[86] C Ringot V Sol M Barriegravere Nm Saad P Bressollier R Granet P CouleaudCl Frochot P Krausz Triazinyl porphyrin-based photoactive cotton fabricspreparation characterization and antibacterial activity Biomacromolecules12 (2011) 1716 httpdxdoiorg101021bm200082d

[87] C Ringot V Sol R Granet P Krausz Porphyrin-grafted cellulose fabric newphotobactericidal material obtained by ldquoClick-Chemistryrdquo reaction MaterLett 63 (2009) 1889 httpdxdoiorg101016jmatlet200906009

[88] R Rychtarikova S Sabata J Hetflejs G Kuncova Composites with photosen-sitive 5101520-tetrakis(N-methylpyridinium-4-yl)porphyrin entrappedinto silica gels J Solgel Sci Technol 61 (2012) 119 httpdxdoiorg101007s10971-011-2600-y

[89] R Rychtaacuterikovaacute S Sabata J Hetflejs G Kuncovaacute Photodynamic efficiency ofporphyrins encapsulated in polysilsesquioxanes Chem Zvesti 66 (2012) 269httpdxdoiorg102478s11696-012-0153-3

[90] MQ Mesquita JCJMDS Menezes SMG Pires MGPMS Neves MMQSimotildees AC Tomeacute JAS Cavaleiro Acirc Cunha AL Daniel-da-Silva A AlmeidaMAF Faustino Pyrrolidine-fused chlorin photosensitizer immobilized onsolid supports for the photoinactivation of Gram negative bacteria Dyes Pigm(2014) httpdxdoiorg101016jdyepig201404025 (in press)

[91] M Magaraggia G Jori M Soncin CL Schofield DA Russell Porphyrinndashsilicamicroparticle conjugates as an efficient tool for the photosensitised disinfec-tion of water contaminated by bacterial pathogens Photochem PhotobiolSci 12 (2013) 2170 httpdxdoiorg101039C3PP50282A

[92] MQ Mesquita JC Menezes MG Neves AC Tome JA Cavaleiro A Cunha AAlmeida S Hackbarth B Roumlder MAF Faustino Photodynamic inactivationof bioluminescent Escherichia coli by neutral and cationic pyrrolidine-fusedchlorins and isobacteriochlorins Bioorg Med Chem Lett 24 (2014) 808httpdxdoiorg101016jbmcl201312097

[93] CMB Carvalho ATPC Gomes SCD Fernandes ACB Prata MA AlmeidaMA Cunha JPC Tomeacute MAF Faustino MGPMS Neves AC Tomeacute JASCavaleiro Z Lin JP Rainho J Rocha Photoinactivation of bacteria in wastew-ater by porphyrins bacterial beta-galactosidase activity and leucine-uptakeas methods to monitor the process J Photochem Photobiol B 88 (2007) 112httpdxdoiorg101016jjphotobiol20070401

[94] AV Vorobey SV Pinchuk Photodamage to spores of Fusariumfungi sensitized by protoporphyrin IX Biophysics 53 (2008) 386httpdxdoiorg101134s0006350908050114

[95] S Wohllebe P Richter D-P Haumlder Chlorophyllin for the control

of Ichthyophthirius multifiliis (Fouquet) Parasitol Res 111 (2012) 729httpdxdoiorg101007s00436-012-2893-

[96] I Banerjee MP Douaisi D Mondal RS Kane Light-activated nanotube-porphyrin conjugates as effective antiviral agents Nanotechnology 23 (2012)105101 httpdxdoiorg1010880957-44842310105101

5 tobiol


[97] T Shiragami J Matsumoto H Inoue M Yasuda Antimony porphyrin com-plexes as visible-light driven photocatalyst J Photochem Photobiol C Rev6 (2005) 227 httpdxdoiorg101016jjphotochemrev200512001

[98] H Yokoi T Shiragami J Hirose T Kawauchi K Hinoue Y Fueda K NobuharaI Akazaki M Yasuda Bactericidal effect of a silica gel-supported porphyri-natoantimony(V) complex under visible light irradiation World J MicrobiolBiotechnol 19 (2003) 559

[99] Z Luksiene N Kurilcik S Jursenas S Radziute V Buda Towards environmen-tally and human friendly insect pest control technologies photosensitizationof leafminer flies Liriomyza bryoniae J Photochem Photobiol B 89 (2007) 15httpdxdoiorg101016jjphotobiol200707001

[100] I Buchovec E Paskeviciute Z Luksiene Photosensitization-based inac-tivation of food pathogen Listeria monocytogenes in vitro and on thesurface of packaging material J Photochem Photobiol B 99 (2010)httpdxdoiorg101016jjphotobiol201001007

[101] Z Luksiene I Buchovec E Paskeviciute Inactivation of several strains ofListeria monocytogenes attached to the surface of packaging material byNa-chlorophyllin-based photosensitization J Photochem Photobiol B 101(2010) 326 httpdxdoiorg101016jjphotobiol201008002

[102] Z Luksiene R Kokstaite P Katauskis V Skakauskas Novel approach foreffective and uniform inactivation of Gram-positive Listeria monocytogenesand Gram-negative Salmonella enterica by photosensitization Food TechnolBiotechnol 51 (2013) 338

[103] Z Luksiene E Paskeviciute Novel approach to the microbial decontaminationof strawberries chlorophyllin-based photosensitization J Appl Microbiol110 (2011) 1274 httpdxdoiorg101111j1365-2672201104986x

[104] TA El-Tayeb MM Gharib AM Al-Gendy Preliminary study to inves-tigate the optimum parameters of using hematoporphyrin IX to con-trol flesh fly (Parasarcophaga argyrostoma) J Entomol 8 (2011) 384httpdxdoiorg103923je2011384390

[105] MC Gomes SM Woranovicz-Barreira MAF Faustino R FernandesMGPMS Neves AC Tome NCM Gomes A Almeida JAS Cavaleiro ACunha JPC Tome Photodynamic inactivation of Penicillium chrysogenumconidia by cationic porphyrins Photochem Photobiol Sci 10 (2011) 1735httpdxdoiorg101039c1pp05174a

[106] I Buchovec R Kokstaite Z Luksiene V Pamedytyte R Makuska Antimi-crobial efficiency of photoactivated chlorophyllinndashchitosan complex ChemTechnol 61 (2012) 54 httpdxdoiorg105755j01ct6132710

[107] I Banerjee D Mondal J Martin RS Kane Photoactivated antimicrobial activ-ity of carbon nanotubendashporphyrin conjugates Langmuir 26 (2010) 17369httpdxdoiorg101021la103298e

[108] C Ringot N Saad R Granet P Bressollier V Sol P Krausz Meso-functionalized aminoporphyrins as efficient agents for photo-antibacterialsurfaces J Porphyr Phthalocya 14 (2010) 925 httpdxdoiorg101142S1088424610002719

[109] T Ben Amor L Bortolotto G Jori Porphyrins and related com-pounds as photoactivatable insecticides 3 Laboratory and field studiesPhotochem Photobiol 71 (2000) 124 httpdxdoiorg1015620031-8655(2000)0710124sippar20co2

[110] E Alves MAF Faustino JPC Tomeacute MGPMS Neves AC Tomeacute JASCavaleiro Acirc Cunha NCM Gomes A Almeida Photodynamic antimi-crobial chemotherapy in aquaculture photoinactivation studies of Vibriofischeri PLoS ONE 6 (2011) e20970 httpdxdoiorg101371journalpone0020970

[111] E Alves JMM Rodrigues MAF Faustino MGPMS Neves JAS Cav-aleiro Z Lin A Cunha MH Nadais JPC Tomeacute A Almeida A newinsight on nanomagnet-porphyrin hybrids for photodynamic inactiva-tion of microorganisms Dyes Pigm 110 (2014) 80 httpdxdoiorg101016jdyepig201405016

[112] J Hemingway BJ Beaty M Rowland TW Scott BL Sharp The innovativevector control consortium improved control of mosquito-borne diseasesTrends Parasitol 22 (2006) 308 httpdxdoiorg101016jpt200605003

[113] K Walker Cost-comparison of DDT and alternative insecticides formalaria control Med Vet Entomol 14 (2000) 345 httpdxdoiorg101046j1365-2915200000262x

[114] M Zaim P Guillet Alternative insecticides an urgent need Trends Parasitol18 (2002) 161 httpdxdoiorg101016S1471-4922(01)02220-6

[115] R Nauen Insecticide resistance in disease vectors of public health importancePest Manage Sci 63 (2007) 628 httpdxdoiorg101002ps1406

[116] A Barbieri Sensibilizadores fluorescentes como larvicidas Action fotody-namica de la luz Riv Malariol 7 (1928) 456

[117] J Robinson Photodynamic insecticides a review of studies on pho-tosensitizing dyes as insect control agents their practical applicationhazards and residues Residue Rev (1983) 69 httpdxdoiorg101007978-1-4612-5569-7 2

[118] CA Rebeiz JA Juvick CC Rebeiz Porphyric insecticides I Concepts andphenomenology Pestic Biochem Physiol 30 (1988) 11

[119] CA Rebeiz JA Juvik CC Rebeiz CE Bouton LT Gut Porphyric insec-ticides 2 110 Phenanthroline a potent porphyric insecticide modulatorPestic Biochem Physiol 36 (1990) 201 httpdxdoiorg1010160048-

3575(90)90011-P

[120] CA Rebeiz A Montazer-Zouhoor IM Mayasich BC Tripathy SMWu CC Rebeiz Photodynamic herbicides Recent developments andmolecular basis of selectivity CRC Crit Rev Plant Sci 6 (1988) 385httpdxdoiorg10108007352688809382256


[121] CA Rebeiz KN Reddy UB Nandihalli J Velu Tetrapyrrole-dependentphotodynamic herbicides Photochem Photobiol 52 (1990) 1099httpdxdoiorg101111j1751-10971990tb08451x

[122] K Snow C Ramsdale Distribution chart for European mosquitoes EuropeanMosquito Bulletin Journal of the European Mosquito Control Association1999 pp 14

[123] Directorate-General of Health from the Portuguese Ministry of HealthMonthly Updates on Outbreak of Dengue in Madeira Situation in07042013 2013 (Internet) Available from httpwwwdgsptcn=683368347243AAAAAAAAAAAA (updated 010913 accessed 021013)

[124] European Commission Regulation EU 11292011 Official Journal of the Euro-pean Union Brussels 2011

[125] FAP Schaffner J Medlock (Eds) European Mosquito Bulletin Journal ofthe European Mosquito Control Association 2013 (Internet) Available fromhttpe-m-borg (accessed 280413)

[126] European Union Food Additives Authorized for Use in Foodstuffs Intendedfor Human Consumption Council Directive 89107EEC Geneva 1989

[127] European Commission Union List of Food Additives Official Journal of theEuropean Union Commission Regulation (EU) No 11292011 Brussels 2011

[128] A Brandis Y Salomon A Scherz Chlorophyll sensitizers in photodynamictherapy in B Grimm R Porra W Ruumldiger H Scheer (Eds) Chlorophylls andBacteriochlorophylls Springer Netherlands 2006 p 461

[129] T Ben Amor L Bortolotto G Jori Porphyrins and related compounds asphotoactivatable insecticides 2 Phototoxic activity of meso-substitutedporphyrins Photochem Photobiol 68 (1998) 314 httpdxdoiorg101111j1751-10971998tb09687x

[130] T Ben Amor G Jori Sunlight-activated insecticides historical backgroundand mechanisms of phototoxic activity Insect Biochem Mol Biol 30 (2000)915 httpdxdoiorg101016S0965-1748(00)00072-2

[131] N Ngwenya E Ncube J Parsons Recent advances in drinking water dis-infection successes and challenges in DM Whitacre (Ed) Reviews ofEnvironmental Contamination and Toxicology Springer New York 2013 p111

[132] S Somani N Ingole I Principal A Ghatkhed Alternative approach to chlori-nation for disinfection of drinking water ndash an overview Int J Adv Eng ResStud 1 (2011) 47

[133] X Qu J Brame Q Li PJJ Alvarez Nanotechnology for a safe and sustainablewater supply enabling integrated water treatment and reuse Acc Chem Res46 (2013) 834 httpdxdoiorg101021ar300029 v

[134] KS Baker RC Smith The role of solar ultraviolet radiation in marineecosystems in J Calkins (Ed) Proceedings of a NATO Conference onthe Role of Ecosystems Held July 28ndash31 1980 in Copenhagen DenmarkNATO Conference Series 4 Marine Sciences Plenum Press New York 1982p 233

[135] A Oliveira A Almeida CMB Carvalho JPC Tomeacute MAF FaustinoMGPMS Neves AC Tomeacute JAS Cavaleiro Acirc Cunha Porphyrinderivatives as photosensitizers for the inactivation of Bacillus cereusendospores J Appl Microbiol 106 (2009) 1986 httpdxdoiorg101111j1365-2672200904168x

[136] L Costa JP Tomeacute MG Neves AC Tomeacute JA Cavaleiro Acirc Cunha MAFaustino A Almeida Susceptibility of non-enveloped DNA-and RNA-typeviruses to photodynamic inactivation Photochem Photobiol Sci 11 (2012)1520 httpdxdoiorg101039C2PP25156F

[137] J Brame PJJ Alvarez Challenges and opportunities in the use of nanotechnol-ogy for water treatment and wastewater reuse in K Korhonen-Kurki M Fox(Eds) Towards New Solutions in Managing Environmental Crisis Proceed-ings of the USA ndash Iran ndash Finland Environmental Workshop 2009 September14ndash15 Haikko Finland Helsinki University Printing House Helsinki 2010 p84

[138] J Theron JA Walker TE Cloete Nanotechnology and water treatmentapplications and emerging opportunities Crit Rev Microbiol 34 (2008) 43httpdxdoiorg10108010408410701710442

[139] J Almeida J Tome G Neves A Tome JAS Cavaleiro L Costa A Cunha MAFFaustino A Almeida Photodynamic inactivation of multidrug-resistant bac-teria in hospital wastewaters influence of residual antibiotics PhotochemPhotobiol Sci 13 (2014) 626 httpdxdoiorg101039c3pp50195g

[140] FC Cabello HP Godfrey A Tomova L Ivanova H Doumllz A Millanao AHBuschmann Antimicrobial use in aquaculture re-examined its relevance toantimicrobial resistance and to animal and human health Environ Microbiol15 (2013) 1917 httpdxdoiorg1011111462-292012134

[141] FC Cabello Heavy use of prophylactic antibiotics in aquaculture agrowing problem for human and animal health and for the environ-ment Environ Microbiol 8 (2006) 1137 httpdxdoiorg101111j1462-2920200601054x

[142] A Almeida Acirc Cunha NC Gomes E Alves L Costa MA Faustino Phagetherapy and photodynamic therapy low environmental impact approachesto inactivate microorganisms in fish farming plants Marine Drugs 7 (2009)268

[143] Z Luksiene Novel approach to control pathogenic and harmful microorgan-isms in non-thermal way photosensitization in IS Boziaris (Ed) Novel FoodPreservation and Microbial Assessment Techniques CRC Press Boca Raton

2014 p 184

[144] K Plaetzer Photodynamic inactivation based on natural photosensitizers forfood decontamination in Eighth International Conference on Porphyrins andPhthalocyanines (ICPP-8) Society of Porphyrins and Phthalocyanines (SPP)Istanbul Turkey 2014

tobiol

[

[

[

[

[

[

[

[

intracellular uptake of the photosensitizer J Appl Microbiol 114 (2013) 36httpdxdoiorg101111jam12018


145] Z Luksiene A Zukauskas Prospects of photosensitization in control ofpathogenic and harmful micro-organisms J Appl Microbiol 107 (2009) 1415httpdxdoiorg101111j1365-2672200904341x

146] S Pelicano Portuguese Company Creates Anti Mosquito Nanoparticle Suc-cess Gadgetcopy 2013 (Internet) Available from httpwwwsuccessgadgetcomnewsempresa-portuguesa-cria-nanoparticula-antimosquitos-1setlanguage=en (accessed 290413)

147] Self-Cleaning Tiles Revigresreg Lab 2013 (Internet) Available fromhttpwwwrevigresptmicrositephpid=4amplg=gb (accessed 040513)

148] O Jirsaacutek F Sanetrniacutek D Lukaacutes V Kotek L Martinovaacute J Chaloupek inventorsCZ Patent 294274 (2003) PCTCZ2004000056 (2004)

149] A Greiner JH Wendorff Electrospinning a fascinating method for thepreparation of ultrathin fibers Angew Chem Int Ed Engl 46 (2007) 5670httpdxdoiorg101002anie200604646

150] S Dancer How do we assess hospital cleaning A proposal for microbiolog-ical standards for surface hygiene in hospitals J Hosp Infect 56 (2004) 10httpdxdoiorg101016jjhin200309017

151] WR Midden SY Wang Singlet oxygen generation for solution kinetics

clean and simple J Am Chem Soc 105 (1983) 4129 httpdxdoiorg101021ja00351a001

152] G Bertoloni B Salvato M DalllsquoAcqua M Vazzoler G Jori Hematoporphyrin-sensitized photoinactivation of Streptococcus faecalis Photochem Photobiol39 (1984) 811 httpdxdoiorg101111j1751-10971984tb08864x


[153] K Komagoe H Kato T Inoue T Katsu Continuous real-time monitoringof cationic porphyrin-induced photodynamic inactivation of bacterial mem-brane functions using electrochemical sensors Photochem Photobiol Sci 10(2011) 1181 httpdxdoiorg101039c0pp00376j

[154] Y Nitzan S Gozhansky Z Malik Effect of photoactivated hematoporphyrinderivative on the viability of Staphylococcus aureus Curr Microbiol 8 (1983)279 httpdxdoiorg101007bf01577728

[155] X Ragagraves M Agut S Nonell Singlet oxygen in Escherichia coli new insights forantimicrobial photodynamic therapy Free Radical Biol Med 49 (2010) 770httpdxdoiorg101016jfreeradbiomed201005027

[156] SA Bezman PA Burtis TPJ Izod MA Thayer Photodynamic inactivationof E coli by Rose Bengal immobilized on polystyrene beads PhotochemPhotobiol 28 (1978) 325 httpdxdoiorg101111j1751-10971978tb07714x

[157] A Preuszlig L Zeugner S Hackbarth MAF Faustino MGPMS Neves JASCavaleiro B Roeder Photoinactivation of Escherichia coli (SURE2) without

[158] T Dahl W RobertMiddenand P Hartman Pure singlet oxygen cytotoxic-ity for bacteria Photochem Photobiol 46 (1987) 345 httpdxdoiorg101111j1751-10971987tb04779x

Potential applications of porphyrins in photodynamic inactivation beyond the medical scope

1 Introduction





3 Applications for domestic industrial and healthcare settings




4 Conclusions

Acknowledgements

References

tobiol


Eliana Alves was born in Porto (Portugal) She receivedher MSc degree in 2007 and her PhD degree in Biol-ogy in 2013 both from the Department of Biology andCESAM of the University of Aveiro (Portugal) During sevenyears her research work was totally dedicated to Photobi-ology namely to microbial photodynamic therapy usingcationic porphyrins as photosensitizers At the momentshe is a Postdoctoral Research Fellow at the Departmentof Chemistry and QOPNA of the University of Aveiro whereshe is acquiring expertise in lipidomics an expanding fieldof lipid analysis through chromatographic and mass spec-trometry techniques

Maria Amparo F Faustino received her doctoraldegree in Chemistry from University of Aveiro in1999 She is currently working as Assistant Profes-sor in the Department of Chemistry University ofAveiro Her current area of research includes synthe-sis and transformation of tetrapyrrolic derivatives andtheir applications mainly environmental applicationshttporcidorg0000-0003-4423-3802

Maria da Grac a PMS Neves is Associated Professor withHabilitation at the Department of Chemistry of the Univer-sity of Aveiro (UA) She obtained her Habilitation and PhDdegree from UA her MSc degree from UMIST Manch-ester (Great Britain) and her BSc degree in Chemistryby University of Lourenc o Marques (Mozambique) Herresearch interests are centered on the synthesis func-tionalization and potential applications of tetrapyrrolicmacrocycles like porphyrins corroles and phthalocya-nines httpsorcidorg0000-0002-7953-8166

Acircngela Cunha is Assistant Professor at the Department ofBiology and researcher of the Center for Environmentaland Marine Studies (CESAM) of the University of Aveiro(Portugal) She received her PhD degree in Biology in2001 at the University of Aveiro and has been workingon environmental microbiology and microbial ecology ofaquatic environments Her recent interests are the studyof the effect of biosurfactants on the development ofmicrobial biofilms and on of photodynamic approaches forthe prevention and control of microorganisms with a par-ticular focus on resistant structures such as endosporesand polimicrobial biofilms

M Helena G A G Nadais is Assistant Professor at theDepartment of Environment and Planning in the Univer-sity of Aveiro (UA) She is a Chemical Engineer holding aMSc in Chemical Engineering (1993) from Instituto Supe-rior Tecnico Technical University of Lisbon Portugal anda PhD (2002) in Environmental Science from the UA Herresearch interests are centered on water treatment andon material and energetic valorization of wastewaters andwastes She has more than 60 publications She has partic-ipated whether as team member or as main investigator invarious scientific research projects and recently submit-ted a patent application She is full member of the researchcenter CESAM (wwwcesampt)

Adelaide Almeida is Assistant Professor at the Depart-ment of Biology from the University of Aveiro (Portugal)where she got her PhD degree in 2001 She is an inte-grated member of the Associated Laboratory Centre forEnvironmental and Marine Sciences (CESAM) In the lastyears she has been involved in the development and

application of alternative methods to the use of antibi-otics such as photodynamic therapy and phage therapyShe has published in these fields and her publications canbe found in httpwwwcesamuaptadelaidealmeida


1 Introduction

Photodynamic therapy refers to the use of a light source (visiblelight of an appropriate wavelength) an oxidizing agent (molecularoxygen O2) and an intermediary agent (named photosensitizer PS)able to absorb and transfer the energy of the light source to molec-ular oxygen leading to the formation of highly cytotoxic species(singlet oxygen [1O2] hydrogen peroxide [H2O2] andor free radi-cals such as superoxide [O2

minusbull] and hydroxyl radical [HObull]) causinga multi-targeted damage and destruction of living tissues [12]The generation of these reactive oxygen species (ROS) can occurvia two mechanisms or pathways known as type I and type IIwhich require the presence of O2 (Fig 1) In the presence of light(h) the photosensitizer in the singlet ground state absorbs a pho-ton affording the excited singlet state Then it can lose energy byreturning to the singlet ground state with fluorescence emission(F) or through an intersystem crossing (ISC) process it can be con-verted in the long-lived triplet state This excited triplet-state PScan decay to ground state by phosphorescence emission (P) or canreact with a substrate namely an electron donor molecule In thiscase the formation of radical ions can occur giving rise to radicalions which react with ground state oxygen (3O2) originating ROS(type I mechanism) Alternatively the excited triplet-state PS cantransfer energy directly to molecular oxygen affording the excitedsinglet state (1O2) (type II mechanism) Both photoprocesses mayoccur simultaneously but type II is in general the predominant oneThe cytotoxic species can cause irreversible damage to proteinsnucleic acids and lipids [34]

The advantage of being a process without a specific cell targetrenders photodynamic inactivation (PDI) effective in the oxidationof different biomolecules with the consequent destruction of sev-eral cell types In fact this methodology has a broad spectrum ofactivity and using the same PS is able to destroy human cells[1] viruses [5] bacteria [6] molds [7] yeasts [8] protozoa [9]helminths [10] and insects [11]

Moreover the ability to structurally tailor the PS as well as tosuccessfully link it to other molecules with a high degree of speci-ficity (eg antibodies enzymes nucleic acids) or to solid supportsgives this therapy a multiplicity of clinical and non-clinical appli-cations

The discovery that positively charged PS could effectivelyinactivate Gram-negative bacteria without the addition of perme-abilizing agents [1213] brought a new impetus to the investigationon the PDI of microorganisms as a new therapeutic modal-ity

The difference in susceptibility between the two types of bac-teria Gram-negative and Gram-positive is explained on the basisof the structural features of their cell wall (Fig 2) Gram-positivebacteria have a cell wall composed of lipoteichoic and teichoicacids organized in multiple layers of peptidoglycan which confersa degree of porosity to bacteria so as to facilitate the anchoringand entry of PS into the cell [1415] In Gram-negative bacte-ria the presence of a complex outer membrane in the cell wallconsisting of phospholipids lipopolysaccharides lipoteichoic acidsand lipoproteins creates an impermeable barrier to antimicrobialagents [1415] The interaction between the cationic PS and theconstituents of the Gram-negative cell wall generates electrostaticinteractions that promote destabilization of the native organiza-tion of the wall allowing the binding and eventual entry of the PSmolecules into the cell [1415] In the case of fungi the cell wallcontains chitin glucans and lipoproteins that represent a barrierwith intermediate permeability in comparison to Gram-positive

and Gram-negative bacteria [16] With regard to viruses envelopedviruses are more easily inactivated than non-enveloped ones butsome studies show that non-enveloped viruses can also be effi-ciently inactivated by the phototoxic action of cationic PS [17]






Singlet oxygen


Type I mechanism

Type II mechanism

h ν

FISC

ICISC

ng pho

bn

ht(p(f

oteon

sTva

t[tgr

wufl(howFlvdrct

P



























ers us

cairo

eifotAcma

ip[dcspAltsipRiA















tobiol


TL






Porphyrin





larvae [46]


ts


No [47]No [505269]



Table 2 (Continued)






Table 2 (Continued)




No [105]


No [445399100102]


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References






Singlet oxygen


Type I mechanism

Type II mechanism

h ν

FISC

ICISC

ng pho

bn

ht(p(f

oteon

sTva

t[tgr

wufl(howFlvdrct

P



























ers us

cairo

eifotAcma

ip[dcspAltsipRiA















tobiol


TL






Porphyrin





larvae [46]


ts


No [47]No [505269]



Table 2 (Continued)






Table 2 (Continued)




No [105]


No [445399100102]


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References







ers us

cairo

eifotAcma

ip[dcspAltsipRiA















tobiol


TL






Porphyrin





larvae [46]


ts


No [47]No [505269]



Table 2 (Continued)






Table 2 (Continued)




No [105]


No [445399100102]


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol


TL






Porphyrin





larvae [46]


ts


No [47]No [505269]



Table 2 (Continued)






Table 2 (Continued)




No [105]


No [445399100102]


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References


Table 2 (Continued)






Table 2 (Continued)




No [105]


No [445399100102]


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References


Table 2 (Continued)




No [105]


No [445399100102]


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References


Table 2 (Continued)


No [78459959]




[(piHdg






F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References


F nsect v

oaou

fl1flaahiiPs[ele

nutDpiUpc6imm













4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

4 tobiol

3wwa

tpo2b(aomidtPtr


teadt























tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol


owdtanamta

tatMa(wicta1wtwwWP[

pLeac1ordlIti























4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

4 tobiol

tmiiTcbGaTl[

tric3

mcipmistea

pfai(uttoswadrrtat

oftdatwt(ttsw

ph





















tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

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4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol

ibta

adidts


























4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

4 tobiol

tfioa

2

amtgoti

ifmmocsvip

hvbotobc

mTAb1itoapfa9hwst

Pi(lat5rtd5p























tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

trt















4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol



aSb(epb
















47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

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4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References


47N(Bb7

pAwfftusavr

e

bullbullbull

bullbull

bull

3s


gmoc


References




[99]


[4981110]




[58103]




materials[82]


[617379]



[7379]





















tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


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4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol

ifii[



n



















5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


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4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

5 tobiol

vmdwtfpl[t



tae(itpi






















tobiol

bpan5rcwwtteTptpt[

c((asadPl


stcwslppbatp

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4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol

bpan5rcwwtteTptpt[

c((asadPl


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4 Conclusions






Acknowledgements


References







5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

5 tobiol



















































comphotosletterpdf


tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol



















































5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

5 tobiol

























3575(90)90011-P


























2014 p 184


tobiol

[

[

[

[

[

[

[

[




















1 Introduction









4 Conclusions

Acknowledgements

References

tobiol

[

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1 Introduction









4 Conclusions

Acknowledgements

References

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