Review Article A Comprehensive Tutorial on In Vitro ...downloads.hindawi.com/journals/bmri/2013/840417.pdfPhotodynamic therapy and PDI require, as multidisci-plinary approaches, the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 840417 17 pageshttpdxdoiorg1011552013840417

Review ArticleA Comprehensive Tutorial on In Vitro Characterization of NewPhotosensitizers for Photodynamic Antitumor Therapy andPhotodynamic Inactivation of Microorganisms

Tobias Kiesslich12 Anita Gollmer3 Tim Maisch3 Mark Berneburg4 and Kristjan Plaetzer5

1 Department of Internal Medicine I Paracelsus Medical UniversitySalzburger Landeskliniken (SALK)Muellner Hauptstrasse 48 5020 Salzburg Austria

2 Institute of Physiology and Pathophysiology Paracelsus Medical University Strubergasse 21 5020 Salzburg Austria3 Department of Dermatology University Hospital Regensburg Franz-Josef-Strauss-Allee 11 93053 Regensburg Germany4Department of Dermatology Eberhard Karls University Liebermeisterstraszlige 25 72076 Tuebingen Germany5 Laboratory of Photodynamic Inactivation of Microorganisms Department of Materials Science and PhysicsUniversity of Salzburg Hellbrunnerstraszlige 34 5020 Salzburg Austria

Correspondence should be addressed to Kristjan Plaetzer kristjanplaetzersbgacat

Received 27 December 2012 Accepted 19 April 2013

Academic Editor Andre Van Wijnen

Copyright copy 2013 Tobias Kiesslich et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In vitro research performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization ofa novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation(PDI) of microorganisms Although many experimental steps of PS testing make use of the wide spectrum of methods readilyemployed in cell biology special aspects of working with photoactive substances such as the autofluorescence of the PS moleculeor the requirement of light protection need to be considered when performing in vitro experiments in PDTPDI This tutorialrepresents a comprehensive collection of operative instructions by which based on photochemical and photophysical propertiesof a PS its uptake into cells the intracellular localization and photodynamic action in both tumor cells and microorganismsnovel photoactive molecules may be characterized for their suitability for PDTPDI Furthermore it shall stimulate the effortsto expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fieldsof applications and motivate scientists of all disciplines to get involved in photodynamic research

1 Introduction

Photodynamic procedures combine three per se harmlesscomponents namely a light-sensitive molecule (the photo-sensitizer PS) non-UV light corresponding to an absorptionpeak of the PS and molecular oxygen to remove harmfulpathogens cells or tissue(s) Within the last decades themost prominent application of this approach photodynamictherapy (PDT) has become a valuable alternative to classi-cal treatment of localized malignant diseases since clinicalstudies prove its effectiveness particularly in early stagetumors The success of PDT is based on three mechanismsby which either alone or in combination the light-inducedand PS-catalyzed overproduction of reactive oxygen species

(ROS) destroys tumors direct tumor cell death damage setto the vasculature and induction of a local inflammationwith a subsequent immune response For some indications(eg skin precancers and cancers) PDT represents a valu-able therapeutic option also due to the excellent cosmeticoutcome Based on the design of new PS (with near infraredabsorption) PS formulations or PSnanoparticle conjugatesas well as technical improvements still new applications ofPDT are regularly identified [1]

Motivated by the achievements of recent PDT researchand justified by the severe health threat that antimicrobialresistance poses to humansrsquo health photodynamic inacti-vation of microorganisms (PDI) has been (re)introducedas revolutionary approach to kill bacteria viruses yeasts

2 BioMed Research International

and parasites The lack of new antibiotics classes combinedwith the propagation of multidrug resistant bacteriafungiand the economic and regulatory challenges thereof haveboosted the research on PDI [2ndash4] As the properties ofideal PS for PDT and PDI differ numerous new substancesespecially synthesized for this promising approach are beingdeveloped in the photodynamic research community andexpand the field of applications (eg to water-borne diseases)[5 6]

Photodynamic therapy and PDI require as multidisci-plinary approaches the tight cooperation of chemists (egfor synthesis of new PS) biologists (for testing new sub-stances) physicists (eg for light dosimetry) and clinicians(for the transfer from the lab bench to the clinical applica-tion) The development of novel PDTPDI applications inbiomedicine and biotechnology is mainly driven by chemistsand biologists The former design new PS with promisingproperties sometimes without the claim to intend the newsubstance for a specific target and the latter test these newdyes in vitro on eukaryotic or prokaryotic cell cultures inorder to estimate the possible field of application of a PSAlthough many experimental steps of PS testing make useof the wide spectrum of methods readily employed in cellbiology some special aspects (eg fluorescence of the PSmolecule or the requirement of subdued light conditions)have need of being considered when doing in vitro experi-ments in PDTPDI Up to date the scientific photodynamiccommunity did neither suggest a standard strategy for PStesting nor does a tutorial on PS testing exist

The aimof this paper is therefore to provide a comprehen-sive overview of experimental strategies and methodsmdashwithout extensive referencing in order to maintain readabil-itymdashby which novel photoactive drugs can be tested invitro for their employment in photodynamic proceduresIt shall represent a suggestion of operative instructions bywhich novel photoactive molecules may be identified assuitable for PDT and PDI based on photochemical andphotophysical properties of a PS its uptake into cells theintracellular localization and photodynamic action in bothtumor cells and microorganismsThis tutorial shall stimulatethe efforts to expand the convincing benefits of photody-namic procedures within both established and new fields ofapplications and motivate (young) scientists to contribute tothe photodynamic research

2 Characterization of the Photochemical andPhotophysical Properties

21 Recording of AbsorptionFluorescence Spectra in VariousSolventsCells The absorptionexcitation wavelength(s) of agiven PS represents a key selection criterion for its applicationin PDT or PDI As a common agreement UV activation ofphotoactive drugs is inadmissible to exclude damage set tothe cellsrsquo genetic information Also light wavelengths whichare absorbed by themajor tissue or cell chromophores shouldbe avoided for excitation of the PS For PDT on solid tumorsthe effective penetration depth of the excitation light highlydepends on the interference with the absorption spectra

of the major tissue chromophores namely (oxy-deoxy-)hemoglobin and melanin as well as water The absorptionspectra of these molecules define the optical window forPDT in tissue which covers the wavelength range of 600ndash850 nm [7] The upper limit of this window is set by theminimal quantum energy which is required for an efficientproduction of singlet oxygen considering thermal loss ofenergy combined with the shifts of the electrons during thephotophysical processes [8] Due to a different compositionof chromophores in microorganisms the lower wavelengthlimit of the optical window defined for PDT applicationsdoes not necessarily apply for photosensitizers employed inPDI

As the photophysical processes in a fluorescing moleculeare dependent on the solvent excitation and emission spectraof a PS should be read in aqueous solutions (buffers) orbiocompatible solvents such as dimethyl sulphoxide (DMSO)or ethanol In order to increase the water solubility ofrather lipophilic PS fetal calfbovine serum (FCBS) or othersolubility enhancers such as polyvinylpyrrolidone [9 10] maybe added for recording of the spectra In very rare cases(eg for photosensitizers showing absorption peaks with anarrow spectral half-width in combination with laser lightillumination) the recording of spectra of the photosensitizerinside the cells might be necessary to assure a wavelengthoverlap of the light used for excitation with the absorptionof the dye Here cells are incubated with the photosensitizerfor a sufficient period of time detached from the surfaceof the cell culture receptacle (eg by trypsinization) andwashed with buffer to remove PS not internalized into cellsFluorescence spectrometers allowing for stirring the solutionof cells inside the cuvettes might be necessary and the spectrahave to be corrected by samples with cells and without PS[11 12]

Additionally the experimenter has to take into accountthat not only the primary PS itself but also secondarymolecules with different absorption resulting from photo-modification of the primary PS may contribute to the overallphotodynamic efficiency [11 13]

The preparation of stock solutions of the photosensitizingagents might prove useful as they might depending onthe chemical stability of the photosensitizer and the stor-age conditions allow for a good experiment-to-experimentreproducibility of the PS concentration in PDT experimentsIf stock solutions are prepared the final concentration ofsolvents other than physiological buffers in the in vitroapplication should be as low as possible (for DMSO andethanol lt1 vv) and the possible cytotoxic effect of thesolvent should be tested by means of a viability assay (seethe following chapter) under conditions identical to thoseused for incubation of the PS with the target cells Storage ofthe PS should be performed according to the manufacturerrsquosrecommendations or may require individual optimizationThe stock solutions of somePSmay be kept frozen (exceptioneg liposomal formulations) Independent of the storageconditions (as solid powder or in solution) the controlmeasurements of the PS spectral properties on a regularbasis are highly recommended to rule out PS decomposi-tion

BioMed Research International 3

22 Monitoring of Singlet Oxygen Production Depending onthe chemical structure of a photosensitizer there are twoalternative kinds of pathways to generate reactive oxygenspecies (ROS) after light activation A type I process involvesthe direct interaction of an excited photosensitizer withsurrounding substrates to generate radicals or radical ionslike hydroxyl radicals (HO∙) and superoxide anions (O

2

∙minus)via charge transfer Whereas in a type II mechanism thegeneration of singlet oxygen (1O

2

) usually takes place bydirect energy transfer from the excited triplet state of thePS to molecular oxygen (see Figure 1) In both cases theinitially generated reactive oxygen species initiate furtheroxidized intermediates at the cell wall cell membrane onpeptides and lipids depending on the localization of thephotosensitizer

The detection of reactive oxygen species is usually shownindirectly by deactivation of ROS using quenchers likesodium azide (type II) histidine mannitol (type I) beta-carotene and superoxide dismutase (type I) In biologicalsystems the lifetime of ROS can be very short (eg few 120583sfor singlet oxygen) [14] Thus the quencherdetection agentsmust be located directly at the site of ROS generation witha sufficient high concentration which can be difficult anda source of ambiguous results However in microorganismsthe transport of such quenchers is rather complicated and thequenchers may not reach the site of ROS generation

Increasingly sophisticated optical techniques have beendeveloped and employed over the last years to both create andmonitor ROS such as 1O

2

in samples that range from liquidsolutions to single living cells [15] 1O

2

is believed to playthe major role in PDTPDI Therefore the following text willfocus on frequently used detection techniques for this typeof ROS Wu et al have recently summarized methods of 1O

2

detection [16] with focus on the recent technical advancesTo investigate the chemistry of 1O

2

several analytical toolsare available to obtain information about the concentrationthe spatial distribution and the temporal behavior of 1O

2

formation including spectrophotometry fluorimetry and(chemi)luminometry

With respect to spectrophotometry over the years thedesign of appropriate probes for 1O

2

has developed signifi-cantly Up until the late 1990s many chemical 1O

2

traps hadbeen reported [17ndash21] Of the trapping molecules used 13-diphenylisobenzofuran (DPBF) has certainly been one of thefavorites [22] 1O

2

rapidly and irreversibly reacts with DPBFto initially yield an endoperoxide which in turn evolves intoother products that do not fluoresce and have absorptionspectra different from that of DPBF The development anduse of DPBF-dependent techniques have recently playeda key role in yielding useful information about 1O

2

in awide variety of systems [23ndash26] 910-diphenylanthracene(DPA) was also a widely used chemical trap for 1O

2

[2728] In both cases as well as for many other moleculesthe fluorophore itself acts as a reactive moiety and changesits photophysical properties upon reaction with 1O

2

Thedecrease in absorbance due to the loss of conjugationhas been used for anthracene derivatives as a quantitativemeasure of the formation of the endoperoxide However

because the detection of 1O2

was based on the measurementof absorbance changes these probes are not highly sensitive

In 1999 Umezawa and coworkers designed and syn-thesized novel fluorometric probes for 1O

2

based on flu-orescence changes in order to improve the sensitivity[29] They developed linked two-component systems calledDPAXs consisting of a fluorophore reporter portion anda 1O2

-reactive anthracene moiety The fluorophore part isbased on fluorescein which is widely used in cell biol-ogy for labeling and sensing [30] In 2001 Tanaka et alreported a rational design strategy for an optimal fluores-cent probe for 1O

2

also based on a fluorescein-anthracenecombination called 9-[2-(3-carboxy-910-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) [30] The mecha-nism these probes are based on is photoinduced electrontransfer (PeT)The experimental approach has been to devisea two-component system comprised of a trapping moietycoupled to a light emitting chromophore Prior to the reactionwith 1O

2

emission from the chromophore is quenched byelectron transfer from the adjacent trapping moiety Uponreaction with 1O

2

however the resultant oxygen adduct isno longer an efficient intramolecular electron donor andlight emission readily occurs from the fluorescent moietyCommercial vendors such as molecular probes also usedthis fluorescein-anthracene combination to develop singletoxygen sensor green (SOSG see Figure 2) [31]

Fluorescent probes are sensitive and can afford highspatial resolution via microscopic imaging [30] The fluores-cent properties of fluorescence probes such as fluorescenceintensity wavelength quantum yield or fluorescence lifetimecan change upon reaction with 1O

2

Two clear advantages ofthis indirect method to detect 1O

2

exist (1) the luminescencequantum efficiency of the optical probe is comparatively large(Φfl sim 10

6 Φph (1O2

) asymp 1) and (2) emission occurs in thevisible region of the spectrumwhere optical detectors are veryefficient However for a number of reasons caution must beexercised when using fluorescent probes such as SOSG Firstit was shown by Ragas et al that SOSG is able to generate1O2

[32] and Gollmer et al have shown that the immediateproduct of the reaction between SOSG and 1O

2

is itselfan efficient 1O

2

photosensitizer [33] Second SOSG appearsto efficiently bind to proteins which in turn can influenceuptake by a cell as well as behavior in the cell [33]Third somefluorescent probes are not selective enough for one particularROS Recently Nakamura et al showed that SOSG reactedwith other ROS such as O

2

∙minus hydrogen peroxide (H2

O2

) andHO∙ resulting in a small increase in the fluorescence response[34] As such incorrect use of fluorescent dyes can yieldmisleading data on yields of photosensitized 1O

2

productionand can also lead to photooxygenation-dependent adverseeffects on the system being investigated

Therefore direct measurements are highly recommendedto detect ROS like 1O

2

via its luminescence at 1270 nm Inthis case photons that are emitted by the excited moleculeitself are detected (Figure 1 right side) An advantage is thatthere is no need for any additional substances which mightbe either toxic or too large for transportation in tissue Thetime-resolved detection technique has been used for (1) the


Abso

rptio

n

1270

nm

O OHO

ClCOOH

COOH

Nonfluorescent

O HO

ClCOOH

COOH

OO

Highlyfluorescent

ISC

PS excitation Energy transfer

Detection

PS fl

uore

scen

ce

Lum

ines

cenc

e

1198781

1198791

O

119878119899

1198780 3O2

1O2

Figure 1 Jablonski diagram for the photosensitized production of 1O2

(left side) and its detection either by direct measurement of the singletoxygen photons at 1270 nm (singlet oxygen luminescence) or indirectly using a fluorescent probe (right side)

HO

ClCOOH

COOH

OO

OHO

ClCOOH

COOH

Internal electrontransfer


SOSGWeakly fluorescent

SOSG-EPHighly fluorescent

O O O

1O2

Figure 2 Formation of the endoperoxide of SOSG (singlet oxygen sensor green) upon reaction of SOSG with 1O2

as an indirect method fordetecting 1O

2

Prior to the reaction with 1O2

internal electron transfer (ET) quenches the fluorescence from the light-emitting chromophoreUpon reaction with 1O

2

and the formation of the endoperoxide electron transfer is precluded and fluorescence is observed

identification of 1O2

(2) measurement of quantum yields of1O2

production in photosensitized processesΦΔ

and (3) thedetermination of rate constants for the interaction of 1O

2

withsubstrates

The 1O2

signal depends on many parameters that areexpressed in the following formula

[1O2

] (119905) =

[1198791

]0

1198961198791Δ

[3O2

]

119896119879

minus 119896119889

(119890minus119896119889119905

minus 119890minus119896119879119905

) (1)

where [1198791

]0

is the concentration of the photosensitizermolecules in the excited 119879

1

state 1198961198791998779

is the rate constant fordeactivation of the 119879

1

state by 1O2

[3O2

] is the concentrationofmolecular oxygen in its ground state 119896

119879

is the rate constantfor all channels of 119879

1

deactivation and 119896119889

is the rate constantfor all channels of 1O

2

deactivationThe time dependence of the 1O

2

signal is used to betterunderstand the interaction of 1O

2

with its environment Ittells one about the kinetics of the production and the decay of1O2

in different environments ranging from polymer systems

to single living cells These direct measurements can reflectthe complex and dynamic morphology of a cell [35ndash39]

The 1O2

luminescence quantum efficiency (ΦPh) is givenby (2)

ΦPh = ΦΔ119896119903120591Δ (2)

where ΦΔ

represents the quantum yield of 1O2

formation 119896119903

the radiative rate constant for the transition 1O2

rarr3O2

and120591Δ

the lifetime of 1O2

However despite the wide spread use of the direct

1270 nm detection it has limitations because of the low 1O2

luminescence (the quantum efficiency is about 10minus5ndash10minus7depending on its environment) [40] and low signal-to-noiseratios which makes the measurement of the 1O

2

signal anontrivial task and limits the effectiveness of this techniquefor many applications especially in the biological field In thepresence of molecules that can physically quench or chemi-cally react with 1O

2

such as water or proteins for example in


a biological cell 120591Δ

will decrease and ΦPh will also decreaseresulting in a low signal-to-noise ratio However for themea-surement of the luminescence signal a very sensitive detec-tion system is availableWith respect to 1O

2

detection the useof the 1270 nm 1O

2

rarr3O2

luminescence in both time andspatially resolved experiments has without doubt been themost beneficial and informative tool Several research groupswere able to detect singlet oxygen luminescence in lipidsand even in living cellsmicroorganisms after incubationwithan exogenous photosensitizer and an optical excitation atdifferent wavelengths [41ndash44]

23 Assessment of Radical Formation In the type I mecha-nism of the photodynamic principle hydrogen-atom abstrac-tion or direct electron-transfer reactions occur between thelight excited state of the photosensitizer and a substrate thatcan be either a biological structure (eg lipids proteinsamino acids or DNA) a solvent or an inanimate surfaceto yield free radicals or radical ions like superoxide rad-icals hydroxyl radical or peroxyl radical [45] The mainmethods for detection of type I-generated radicals includescavenging molecules such mannitol histidine and NN-dimethyl-p-phenylenediamine (DMPD) as well as the totalradical-trapping antioxidant parameter method (TRAP) orthe oxygen-radical absorbance capacity (ORAC) method Atpresent there is a need for more specific assays due to thelower specificity of these scavenging methods Overall type Ireactions become more important at low oxygen concentra-tions like inside of biofilms or in more polar environments[46]

24 Determination of the PhotostabilityBleaching Due tolight exposure of a photosensitizer the absorption and flu-orescence properties of the photosensitizer itself can changewhich indicates that photodegradation andor photoproductformation appeared which results in a decreased photo-stability [47 48] Such changes can be evidenced by theappearance of new absorption bands within the specificabsorption spectra of the photosensitizer [49] Degradationcan be monitored by the decrease of maxima absorptionpeak of the photosensitizer Changes of the characteristicabsorption spectra of a given photosensitizer depend on thewavelength of the illumination light shorter wavelengthsare more effective than longer wavelengths [50] If thephotosensitizer is bleached too rapidly either successfulinactivation of microorganisms or tumor destruction willnot be completed once the minimal inhibitory concentrationof the nondegraded photosensitizer in the infectedtumortissue is deceeded upon illumination [51] On the other handphotobleaching can be an advantage regarding avoiding anoverall skin photosensitivity which is one of the main sideeffects in patients treated with PDT [1 52] Furthermoreit is unalterable to assure the nontoxicity of photodegradedproducts of the photosensitizer Overall knowledge of pho-todegradation as well as effects of photoproducts generatedupon illumination is important to develop an appropriatedosimetry for each photosensitizer in antimicrobial photo-dynamic inactivation or in antitumor PDT [53]

25 Positive Charge and Molecular Weight of a Given Pho-tosensitizer A must-have of a successful PS for PDI isa positive charge because bacteria are charged negativelydue to their cell wall composition and meso-substitutedbut negatively charged porphyrins have not shown toxicityagainst Gram(minus) bacteria [54 55] Furthermore hydrophiliccompounds (less than 600ndash700Da eg forE coli) can diffuseonly through the outer membrane via porins which act as avery effective permeability barrier making Gram(minus) bacterialess susceptible as Gram(+) [56]Therefore porphyrin-basedphotosensitizers like TMPyP with a molecular weight higherthan 500Da cannot diffuse through these porin channels Asa consequence ROS can be generated only at the cell wall areaof Gram(minus) bacteria Another important observation thathas been made about positive-charged cationic antimicrobialphotosensitizers concerns their selectivity for microbial cellscompared to host mammalian cells [57] It is thought thatcationic molecules are only slowly taken up by host cells bythe process of endocytosis while their uptake into bacteriais relatively rapid If illumination is performed within shortintervals after PS application (minutes) the PDT-mediateddamage to host tissue will be minimized

3 PS Uptake Kinetics Dark Cytotoxicity andIntracellular Localization in Tumor Cells

This section describes experimental approaches for initialtumor cell-based characterization of new PS including cel-lular pharmacodynamics cytotoxic effects of the PS in theabsence of light and the intracellular localization of the PSFinally the assessment of the penetration depth in an ex vivoporcine skin model is described

31 PS UptakeRelease Kinetics Measurement of the uptakeof a new PS drug into cancer cells provides information onthe (kinetics of) interaction and membrane transport char-acteristics of the drug and enables a first rough estimationof the drug-to-light intervalmdashas a basis for verification inthe subsequent preclinical validation of the PS The method-ological approaches described here make use of the inherentfluorescence properties of the PS and allow for either absolutequantification of the drug amount bound respective taken upby cells or a relative quantification mainly for example forestimation of time-dependent course of PS uptake

By the nature of the assay format experiments usingmicroplates with 96 wells as the most commonly employedformat allow for time-efficient and multiparametric testingof several variables possibly influencing the uptake char-acteristics A first approach involves incubation of cancercells cultured in 96-well microplates followed by a fixedincubation period (eg 10ndash24 hrs) with a dilution series ofthe PS in the appropriate cell culturemedium and subsequentdetermination of the PS-related fluorescence signal Thesimplest procedure involves washing the cell cultures afterthe incubation and lysis of the cells by detergents such asSDS (sodium dodecyl sulphate) followed by fluorescencemeasurement in a microplate fluorimeter Control experi-ments should be performed to exclude altered fluorescence


characteristics of the PS in the presence of such cell lysisreagents Besides the plasma membrane permeability of thegiven PS several additional factors may influence the cellularuptake including interaction of the PS with (i) constituents offetal calfbovine serum (FCSFBS [58 59]) and with (ii) thecell culture plasticmaterial [60 61]Thefirst parameter can beassessed by using appropriate concentrations of FCS rangingfrom zero to the standard concentration used for routinecultivation of the respective cell type (eg 5ndash15 vv FCSfor numerous cancer cell lines) Such data are importantas similar serum constituents are present in human bloodplasma which may influence the tissue distribution of PSdrugs after systemic administration The second parametermay significantly influence the results obtained from thedescribed simple incubation-lysis-measurement approach asespecially lipophilic PSs may attach to a considerable amountto the cell culture plastic surface [60] As we have demon-strated in a recent study ([61] this issue) the surface-adheredPS in microplates without cells can even exert considerablephototoxic effects after addition of PS-free cells in a rangesimilar to the usual protocol where cells are seeded first andthe PS is added subsequently Therefore appropriate controlexperiments and controls samples need to be included toestimate the amount of PS which is bound by the microplateplastic and which might significantly contribute to the PSfluorescence signal measured after PS incubation and directlysis of the cells In case of a rather lipophilic PS such false-positive fluorescence signals can be avoided by detachingthe cells from the culture receptacle (eg trypsinization orEDTA treatment) transfer to new tubeswells followed by celllysis and fluorescence detection Regardless of whether cell-bound PS fluorescence is measured directly in the microplatewells or after detachment and transfer this approach can bedesigned to allow for absolute quantification of the amount ofcell-bound PS if appropriate cell-free PS dilutions series aresimultaneously measured

An alternative approach is based on fluorescence-activated cell sorting (FACS) analyses based on single-cellanalysis of the cellular fluorescence in a flow cytometer Assuch protocols involve detachment of cells prior to analysisno interference with the measured signal from PS moleculesadhered to the plastic material is expected On the otherhand this approach is only suitable for relative quantificationand each sample usually requires manual work (eg for celldetachment transfer washing steps) implicating a reducednumber of processable samples compared to the microplateformat

Both experimental approaches can be used to charac-terize the dose-dependent PS uptake by cancer cells byidentification of the range of PS concentrations which resultin a measurable PS fluorescencemdashthat is the minimumconcentration as well as a concentration threshold abovewhich a saturation of PS boundtaken up by the cancer cellsoccurs With both methods the influence of the presence ofserum constituents as well as for example the influence ofthe chemical PS formulation can be assessed Subsequentlythe time-dependent uptake studies can be performed toinvestigate the kinetics of PS uptake into cells As mentionedpreviously these data may give first information on the

appropriate drug-to-light interval and allow for definition ofan incubation period for subsequent in vivo experimenta-tion Particularly for time-dependent experiments involvingincubation times in the range of several hours up to daysit is important to correct for the cell number (biomass)present at the individual time points This can be achievedfor example by measurement of the total protein content(eg colorimetric assays such as Bradford bicinchoninic acidassays) of the cell sample as a surrogate parameter for theactual cell number Clearly in highly proliferating cell typesthe cell numbermay influence the amount of PS boundtakenup by the individual cellmdashespecially at lowPS concentrations

The release of PS by cellsmdashthat is transport of the PSmolecules out of the cellmdashis an important parameter asit partly determines the period of photosensitivity in theclinical application that is a PS drug which is rather rapidlyexported from cells is likely to bring about a reduced timeperiod necessary for the patient to readapt to normal daylightconditions PS release experiments can be performed usingboth of the previously mentioned approaches and additionalparameters such as the presence of FCS in the culturemedium can be tested for their influence on the releasekinetics In our hands the FACS-based approach seemsmorereliable for this purpose as its single-cell measurement mayallow for more accurate determination of the residual cell-bound PS (resp the amount of PS released from cells) thanif it is summarily measured in lysed cells After the PSincubation period PS release experiments involve carefulwashing of the cell cultures to remove unbound PSmoleculesand subsequent incubation in PS-free cell culturemedium Ingeneral time periods ranging from for example 10 to 2448hours should serve as valid starting points for analysis of thePS release

32 PS Dark Toxicity The general requirements for an opti-mal photosensitizing agent include low dark toxicity that isnegligible cytotoxicity in the absence of lightThis ensures thevalidity of the dual-specificity ideal of antitumor PDT namelythat both via tumor cell (semi)selective enrichment of thePS and confinement of the illuminated area by appropriatedesign of the light source the cytotoxic action of PDT islimited to the cancerous tissue while sparing adjacent healthycells [62] Experimental assessment of the dark toxicityinvolves incubation of the cells with a PS dilution seriesinitially according to the incubation parameters establishedin the PS uptake experiments described in Section 31Probably the establishment of optimal parameters (egincubation time PS concentration media composition andcell density) may require that experiments on PS uptakecharacteristics and dark toxicity are performed in parallelIn general a large array of appropriate assays is availablefor viability analyses in in vitro cell culture samples Theseassays make use of either measurement of (i) metabolicparameters (activity of metabolic enzyme) as a surrogatereadout for the cellrsquos viability (ii) biochemical and mor-phological changes during apoptosis (iii) proliferation ratesof cells andor (iv) viable cell number employing specificmembrane-impermeable dyes to exclude dead (leaky) cells


Table 1 Tests for cell viability and cell death modes

Assay type Test Measured parameter Signal Instrument Microplatea ReferencesbABS FI Lumi

Metabolicenzyme(s)

MTT XTT WST Mitochondrial enzymes X Microplate reader Yes [77ndash79]

ResazurinCellular dehydrogenaseenzymes andcytochromes

X Microplate reader Yes [80]

Metabolites ATP Intracellular ATP X Microplate reader Yes [81]

Apoptotic changes

Caspase activation Apoptosis-specificproteases X X X Western blot No [77]

Microplate reader Yes [75]

Nuclear fragmentation Chromatin condensationand fragmentation X

Fluorescencemicroscope Yes [82]

Flow cytometer No [75]

DNA ladder DNA cleavage resultingin multiples of 180 Bp X Gel electrophoresis No [83]

Membrane blebbing Characteristic apoptoticbodies X Phase contrast

microscope Yes [67]

PARP cleavage poly-ADP ribosepolymerase cleavage X X Western blot No [77]

Annexin VMembraneexternalization ofAnnexin V

X Flow cytometer No [77]Fluorescencemicroscope Yes [77]

Cyt-c release Mitochondrial cyt-crelease X X Western blot No [77]

ΔΨ

Mitochondrialmembrane potentialbreakdown

X Flow cytometer No [67 84]Fluorescencemicroscope Yes [85]

Cell proliferation3H thymidine DNA incorporation of

3H thymidineBrdUX Scintillation counter Yes [86]

BrdU Microplate reader Yes [87 88]Cell number Direct cell number X Flow cytometer NoABS absorbanceATP adenosine-51015840-triphosphate BrdU 5-bromo-21015840-deoxyuridine caspase cysteine-dependent aspartate-directed proteases FI fluorescenceintensity MTT 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide Lumi luminescence PARP poly(ADP-ribose) polymerase WST water-soluble tetrazolium salt XTT 23-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilideaAssay suitable for use with microplates (yesno)bMethodological references or exemplary studies using the respective test in the context of in vitro PDT

For determination of dark toxicity of a PS in a given cell typeusually only the overall effect on viability or proliferation isinteresting More detailed analysis of the modes of cell deathis rather important for the investigation of the light-inducedeffects of the PS (see Section 4) Anticipatorily these tests arelisted here in Table 1 including a superficial appraisal of theirvarious strengths

For the particular assessment of dark toxicity classicalviability tests based on measurement of the activity ofmetabolic enzymes may be most efficient since these testscan be performed in microplates implicating the possibilityof multiparametric testing including technical replicates foreach sample

33 Intracellular PS Localization Following establishment ofthe overall PS uptake characteristics and the PSrsquos dark toxicitya subsequent experimental step involves the determination of

the intracellular localization and enrichment of the photosen-sitizing drug Again for this purpose the inherent fluorescentproperties of the PS are used

Provided the microscope setup is equipped with theappropriate filter sets first superficial information on theintracellular distribution can be obtained from conven-tional fluorescence microscopy With this approach generalstatements such as preferential localization in the cyto-plasm plasma membrane or the perinuclear region canbe obtained For more detailed analysis the use of specificorganelle-localizing dyes (ldquoorganelle trackersrdquo) is recom-mended Table 2 provides a list (not complete) of fluorescencedyes that might be used for analysis of colocalization with thePS investigated The choice of a particular dye depends onits fluorescence spectrum which should not overlap with theemission wavelength of the PS Particularly if the localizationof the PS is not confined to one clearly identifiable structure


Table 2 Fluorescent probes for cellular organelle counterstaining [89]

Organellecell structure Fluorescent dye(s)

Mitochondria TMRM TMRE rhodamine 123 tetramethylrosamine mitotrackers nonyl acridine orangecarbocyanines dual-emission dyes (JC-1 JC-9)

Endoplasmic Reticulum 33-dihexyloxacarbocyanine iodide [DiOC6(3)] ER-TrackerNucleus DAPI Hoechst-33342 propidium iodide SYTO dyesCytoplasm Calcein AMGolgi apparatus Fluorescent labeled lectinsLysosomes LysoTrackerCell membrane CellTrackerDAPI 410158406-diamidino-2-phenylindole TMRE tetramethylrhodamine ethyl ester TMRM tetramethylrhodamine methyl ester

the use of confocal fluorescence microscopy which providesincreased spatial resolution may be helpful An alternativeapproach for quantification of the PSrsquos intracellular local-ization could involve organelle-specific fractionation forexample by centrifugation techniques and analysis of theorganelle-bound PS fluorescencemdashthis approach is moretime-consuming and may require more extensive optimiza-tion for the particular cell type

34 Assessment of the Penetration Depth of the PS inPorcine Skin After a first positive prescreening of new devel-oped photosensitizers with photodynamic activity againstmicroorganisms in suspension in vitro the next challenge inantimicrobial photodynamic inactivation is to find appropri-ate parameters (eg light dose and incubation time) to inac-tivate relevant key pathogens without harming surroundingtissue in vivoex vivo Therefore penetration and localizationof the given photosensitizers must be investigated usingan ex vivo skin model Recently it could be shown thatan ex vivo porcine skin model can be used because it isproposed as a good test model for human skin based onmany similarities regarding physiological histological andpermeability properties [63] Restriction of a photosensitizerto the stratum corneum without accumulation in deeperparts of the epidermis or dermis might be useful regardinga successful decolonization of pathogens on intact skin[64] Recently it could be shown that localization of thephotosensitizer TMPyP in a water-ethanol formulation wasrestricted to the skin surface only [64] However agents witha molecular weight of gt500Da exhibit a low permeabilitythrough the stratum corneum The molecular weight ofTMPyP is 6822 gsdotmolminus1 (without counterions)Therefore themolecular weight of drugs which are used in transdermaldrug-delivery systems is well belowlt500Da [65] To enhancepenetration through the stratum corneum various formula-tions are available that contain supplements (DMSO alcoholpyrrolidones) which exhibit penetration enhancing activities[66] Overall the main targets are superficial and localizedinfections These areas are readily accessible for the topicalapplication of PS and light neither harming the surroundingtissue nor disturbing the resident microbial flora

4 Photodynamic Action in Tumor Cells

This section describes experimental approaches for charac-terization of the cytotoxic action induced by light includinganalysis of overall viability IC

50

values and specifically thediscrimination of the cell death modes induced by PDT

41 Analysis of Tumor Cell Viability Changes after PDT Afterhaving optimized the incubation parameters (concentrationincubation time and media composition) as described inSections 31 and 32 one can proceed in analysis of thephotoinduced cytotoxic effects of a new PS Using adherentcancer cells (cell lines) the fastest approach is to use amicroplate assay based on the activity of metabolic enzymessuch as MTT or the resazurin assay both of which arequick cheap in terms of reagents and easily established (seeTable 1 for an overview) Similar to such assays determi-nation of intracellular ATP gives a reliable estimation ofthe amount of viable cells after a cytotoxic treatment asthe intracellular concentration of this metabolite (as is theactivity of core metabolic enzymes) is assumed to be held in atight (millimolar) range in viable cells Therefore the overallamount of ATP or the enzyme activity in a population ofcells is supposed to represent the overall viable cell numberImportant to keep in mind is the fact that cells undergoingapoptosis (active cell death) maintain considerable levels ofmetabolic activity respective ATP in order to perform theenergy-requiring steps during the apoptotic cascade [67ndash72]Therefore for all of the mentioned assays the time point toperform the test should be chosen in a way so that apoptoticcells do no longer contribute to the assay readoutThis mightinclude establishment for each individual cell line in orderto determine the time point after treatment where apoptoticcells have finished the cell death programandhave undergonesecondary necrosis This time period may be in the range of24ndash48 hrs after treatment for most cancer cell lines

Besides PDT-treated cell samples each particular exper-iment on overall cell viability should include the followingcontrol samplesmdashagain most easily to be realized in the(96-well) microplate format untreated control (UTC) darkcontrol (DC) light-only control (LOC) the treated samples


and appropriate blank wells required for the assayrsquos blanksubtraction In our experience at least triplicate wells shouldbe included for each of the listed sample types Treated sam-ples are incubated with the PS (as established in Section 31to result in a measurable cellular enrichment of the PS) Asillustrated in Figure 3 subsequent illumination conditionscan be designed in two ways to get an overall impressionon the viability changes following PDT treatment (i) aconstant PS concentration is employed for all treated samplesaccompanied by illumination with different light fluences(Jsdotcmminus2) or (ii) incubation with different concentrations ofthe PS (ie a dilution series) followed by illumination witha constant light fluence Particularly when different lightfluences are applied to individual rows of the microplatewells (as in Figure 3 approach (i)) microplates with clearwell bottoms and black walls should be employed to avoidactivation light crosstalk between the rows of wells duringillumination

Similar to determination of the dark cytotoxicity of thePS comprehensive characterization of the PDT in vitromodelsystem should addressmdashor exclude in most casesmdashpossiblecytotoxic effects of the illumination itself For this purposecells are seeded and incubated in the samemedium as usuallyused for PS incubation but without the PS followed byillumination with different light fluences As mentioned formost applications such a control experiment is to rule outpossible effects of the illumination itself on the viability orproliferation rate of the cells

All the mentioned experimental approaches should beaccompanied prior to the particular assay by routine controlobservation in a conventional light microscope In mostcell lines cytotoxic effects can be readily identified at thislevel by observation of rounding of cells (apoptosis probablyincluding classical apoptotic bodies) and detachment of cells(apoptosis andor necrosis at later time points) Such visualcontrol may help interpretation of the results gained fromassays measuring metabolic enzymes or metabolites as asurrogate parameter of cell viability and help rule out false-positively or -negatively highlow signals

42 Calculation of the IC50

Values Thementioned analyses ofdark toxicity and light-induced cytotoxicity (using differentPS concentrations and a constant light dose) can be usedto calculate a modified IC

50

value This parameter is usuallyreferred to as the half-maximal inhibitory concentration ofan inhibitor in for example enzyme inhibition experimentsIn the context of PDT the IC

50

value is calculated by divisionof the concentration required for 50 cell killing in the dark(lethal dose (LD

50dark)) and the concentration required for50 cell killing following illumination of PS-incubated cellsamples (LD

50PDT) [74] The IC50

value thus measures therelation between the cytotoxic effects of the PS in the darkand following photoactivation a higher IC

50

is indicative of alow dark toxicity or a particular high cytotoxic efficiency afterillumination [75] By its nature use of this parameter makesonly sense for direct comparison between two or more PSsin the same cell model and under comparable illuminationconditions [75 76]

43 Analysis of the Cell Death Mode Further in-depth anal-ysis of the cytotoxic action of a PS following illuminationinvolves the discrimination between essentially three modesof cytotoxicity that is inhibition of proliferation inductionof apoptosis and third induction of necrotic cell death Verylow PDT doses may also cause increased cell proliferationresulting in increased cell viability signals andor cell num-bers As discussed recently the mode of action of PDT canusually cause all the mentioned effectsmdashin a dose-dependentmanner as illustrated in Figure 4 This is in contrast tochemotherapeutic agents or radiation which preferentiallycauses apoptosis as the underlying cytotoxic effect [1 7990]

For rapid and initial analysis of the cellrsquos response interms of proliferation apoptosis and necrosis our group hasdeveloped a simple and versatile assay based on microplateassays analyzing metabolic enzymes [67 70] This proce-dure makes use of the fact that cells undergoing apoptosis(ie active cell death) require functioning energy supply interms of intracellular ATP accompanied by approximatelynormal activity of (catabolic) pathways whose enzymes arethose measured in the MTT test for example As shownin Figure 5 this approach employs standard viability testsbased on metabolic surrogate parameters (eg MTT ATPresazurin) and involves measurement at two different timepoints following PDT treatment a first measurement is takenat an ldquoearlyrdquo time point where cells undergoing apoptosisstill retain their metabolic activity A second reading is takenat a ldquolaterdquo time point where apoptosis has been completedand these cells have converted to secondary necrosis dueto the absence of phagocytizing cells in the cell culturesetting In contrast to apoptotic cells which maintain theirmetabolic activity until the late steps of the apoptotic pro-gram necrotic cells are characterized by a rapid breakdownof the plasmamembrane integrity metabolic hemodynamicsand a leakage of intracellular material in the extracellularspace [91]

As shown in Figure 5 the different signals between earlyand late readings can be used for a first discriminationbetween induction of proliferation apoptosis or necrosisin the in vitro setting This approach clearly works withsum signals therefore mixed populations of for exampleapoptotic and necrotic cells cannot be quantified in absoluteterms However the 96-well microplate formatmdashon the otherhandmdashallows for rapid testing of for example ten differenttreatment conditionsThis assay variant has been successfullyused in previous publications with either the MTT assay[67 70] or the fluorescent resazurin assay [82]

Another simple test for discrimination of whether areduced viability signal is caused by direct cytotoxicity(apoptosis or necrosis) or by inhibition of proliferation alsoemploys metabolic viability tests such as the MTT assayFor this purpose multiple readings at different time pointsfollowing illumination are performed The viability signalsobtained at each time point are related to the initial (119905 =0 hrs) value and the resulting temporal dynamics of thesignal for each treatment condition can be evaluated asfollows a decrease below the initial value can be interpretedas a direct cytotoxic effect as the absolute viability signal


Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12

(i) Light fluence constant PS(ii) [PS] constant light fluence

Viability testeg MTT ATP

resazurin Treated samples

Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s

Light dose 1Light dose 2

(i)

(ii)

PS conc 1 orlight dose 1


Nolight

1 2 3 4 5 6 7 8 9

Light fluence (J middotcmminus2)

PS concentration (molmiddotLminus1)

Figure 3 Experimental variants for analysis of overall tumor cell viability following PDT Under constant PS conditions or constant lightfluence the light fluence or the PS concentration can be varied to obtain initial information on the phototoxic effects of a particular PS DCdark controls (PS without light) PS photosensitizer UTC untreated control (no PS no light)

PDT dose increasing PS conclight fluence

Cel

lula

r res

pons

e

survival necrosisapoptosisMainly Mainly Mainly

Figure 4 Dose-dependent transition between cellular responsesfollowing PDT Abbreviations PS photosensitizer Modified from[73]

decreases A constant viability signal (in the range of theinitial value) indicates inhibition of proliferation whereasa signal increasing relative to the initial value (similar tountreated controls in most cases) indicates proliferation

Clearly as a sum measurement this test design cannotdiscriminate between the modes of cytotoxicity in absoluteterms (ie on the single-cell level) However it may assist inthe interpretation in a situation where the endpoint viabilitymeasurement (eg 24 hrs pi) indicates a viability signalsmaller than the untreated controls since this reduction couldbe solely attributed to growth inhibition without any apop-tosisnecrosis induction that is direct cytotoxicity Assaysdirectly measuring the proliferation rate are classically basedon DNA incorporation of nucleotide analogues such as 3H-thymidine or bromodeoxyuridine (BrdU) Incorporation ofthe first can be measured via scintigraphy whereas the latteris detected by BrdU-specific immunostaining Both methodsallow direct assessment of the proliferation rate of cells butshould be accompanied by the mentioned viability tests forunequivocal interpretation

After having superficially determined how the cellpopulation responds to a photodynamic treatment (sur-vivalproliferation direct cytotoxicityreduced viability) onemay proceed to in-depth characterization of the specificmode of cell death in case of a cytotoxic PDT regimenThis might be relevant to elucidate the detailed mechanism


Table 3 Cell-based assays for discrimination and quantification of cell death modes

Cellularbiochemical event Methoda Assay platform Commentb

DNA degradation

(i) Detection of ldquoDNA laddersrdquothat is multiples of 185 bp Gel electrophoresis Semiquantitative

(ii) TUNEL FM FACS Semi-quantitative

(iii) COMET Single cell gel electrophoresis Semi-quantitative

(iv) SubG1 (cell cycle analysis) FACS Quantitative

Nuclear fragmentationFor example DAPIHoechst-33342 DNA-stainednuclei

FM Quantitative

Membrane blebbing Morphological changes Phase contrast LM Semi-quantitative

Caspase activationFluorometricluminometricdetection of cleavage of artificialcaspase substrates

Microplate reader FM FACS Quantitative single-cell analysisvia FACS

PSer exposure Antibody staining FM FACS Quantitative single-cell analysisvia FACS

Mitochondrial cyt-c release Subcellular fractionation andimmunodetection Western blotting Semi-quantitative

Mitochondrial ΔΨ breakdown Fluorochrome-based assessmentof mitochondrial ΔΨ FM FACS

Semi-quantitative (within cells)quantitative for comparisonbetween cell populations

Membrane integrity release ofintracellular materialc

(i) Detection ofnecrosis-associated plasmamembrane breakdown via PIstaining

FM FACS Quantitative single-cell analysisvia FACS

(ii) Biochemical assay for LDHenzyme release from necroticcells

Microplate reader Quantitative

Cyt-c cytochrome c DAPI 410158406-diamidino-2-phenylindole ΔΨ mitochondrial membrane potential FACS fluorescence-activated cell sorter FMfluorescence microscopy LM light microscopy PI propidium iodide PSer phosphatidylserine TUNEL terminal deoxynucleotidyl transferase-mediateddUTP nick end labelingaSelection of methods is focused on in vitro experimentation (cell culture)bBased on the authorrsquos experiencecThese methods address specific necrosis-associated cellular changes

of a particular PS or PDT regimen and on the otherhand might have implications for overall therapeutic effectby induction of diverse immune system-related responses[92 93] In this brief discussion we focus on the classicalways of cell demisemdashexcluding autophagy which is alsoconsidered in recent reports to contribute to PDT-inducedcytotoxicity [94] (for an methodological overview see [95])Table 3 lists the most common methods and assays toaddress whether cells and cell populations undergo apoptosis(activeldquoprogrammedrdquo cell death) andor necrosis (passivecell death) (further reading [96 97]) As commented inTable 3 the variety of methods differs with respect to theirquantitative or semiquantitative results that is whether apercentage of cells undergoing apoptosis can be determinedusing the particular method Furthermore the assays differwith respect to price as well as prerequisites regardinginstrumentation and time required Furthermore some ofthe listed approaches may depend on whether the cell(or cell line) studied shows the morphologicalbiochemicalfeature addressed by the particular test A comprehensiveweighting of the various methods is beyond the scope of

this sectionmdashuseful advice in our opinion includes the fol-lowing aspects (i) appropriatemdashprobably non-PDT-treated-control samples (ie 100 apoptoticnecrotic cells) help tovalidate the method and make the results obtained for PDT-treated samples more reliable and expressive (ii) wheneverpossible a population-basedmethod should be accompaniedby single-cell analysis(es) to gain information about the cellportion affected (iii) all indirect (non-microscopy-based)assays should be accompanied by simple (phase contrast)light microscopy to allow comparison with the sometimesquite obvious overall cellular responses (iv) the timingwhen to use individual methods may need optimizationfor each cell model (and treatment protocol) as some ofthe cellularbiochemical events listed in Table 3 occur earlyversus rather late following the PDT treatment and (v) PDTtreatments may cause mixed population consisting of bothapoptotic and necrotic subpopulation of cells in a givensample ([73] see also Figure 4)Themethods listed in Table 3comprise assays specifically focusing on in vitro experimen-tation using cells (cell lines) in culture for specific methodsto investigate the occurrence and extent of apoptosisnecrosis


PDT dose increasing PS conclight fluence(a)

(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010

0PDT dose increasing PS conclight fluence

Difference curve

Early reading

Late reading

Survivalproliferation

Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0

Figure 5 Discrimination of the cellular responses towards PDTMeasurement of the overall viability at early versus late time pointsfollowing illumination (a) allows for calculation of a difference curve(b) and estimation of the dose ranges which predominantly inducecellular survival apoptosis and necrosis (c) PS photosensitizer Fordetails on interpretation see text

in situ (tissue sections) the reader is kindly referred to recentmethodological overviews [98 99]

5 Characterization for PhotodynamicInactivation of Eukaryotic andProkaryotic Microorganisms

Ideally a wide spectrum of antimicrobial action on bacteriafungi (yeasts) and protozoa should be achieved with agiven PSPDI protocol The primary readouts should focusfirst on photodynamic efficacy in suspensions followed bybiofilm inactivation (monospecies and polyspecies) in vitroLater on ex vivo and animal studies should be consideredto demonstrate a photodynamic killing efficacy of ge3 log

10

steps (ge999 reduction of viable microorganisms) Such

a reduction of viable microorganisms must be achieved tostate that an antimicrobial effect is possible Furthermore theefficacy should be independent of the antibioticantifungalresistance pattern of the investigated microbial strains Fromthis point of view a selection of photodynamic-resistantmicroorganisms should be absent after multiple sublethaltreatments conditions Due to the regulatory affairs to getapproval by the FDA or the European Health Authoritiesmutagenicity must be excluded Appropriate formulationsmust be developed allowing an easy and specific delivery ofthe given photosensitizer to the infected area

51 Assessment of Cytotoxicity in the Dark and PhototoxicityBased on CFU Counting The American Society of Micro-biology has decreed that for any technique to be calledldquoantibacterialrdquo or ldquoantimicrobialrdquo at the very least 3 log

10

ofCFU (999) need to be killed Furthermore based on theguideline for hand hygiene in health-care settings aminimumof 5 log

10

reduction of viable counts of microorganisms mustbe achieved for a successful disinfection [100] Survival ofviable bacteria must be determined by the colony-formingassay After overnight incubation colonies are counted andviable pathogen concentration is expressed as CFUmL usinga logarithmic scale Furthermore no cytotoxic effects inthe dark of both the given photosensitizer itself and ofthe possible photoproducts formed after illumination shouldbe demonstrated either against the pathogen itself or theeukaryotic cells

52 Addition of Cell Wall-Permeabilizing Agents In case thatthe given photosensitizer is not efficient enough (less than3 log10

steps of CFUmL reduction) to kill relevant pathogensupon illumination addition of cell wall-permeabilizing agentsmight be useful to enhance the photodynamic efficacy Froma clinical point of view metal chelators like EDTA might beuseful to cause a disorganization of lipid structures increasingthe permeability of the outer membrane of Gram(minus) bacteria[54 101] EDTA solutions might be useful because it iswell established in dentistry as it has been commonly usedas a detergent for the removal of smear layers Anotherpermeabilizing agent is polymyxin B nonapeptide which hasdemonstrated a porphyrin-based photodynamic enhance-ment [55]

53 Determination of the Efficiency towards Bacterial BiofilmsThe natural behavior of microorganisms is to grow as abiofilm rather than as free-floating cells It is generallyaccepted that biofilms represent the leading cause of micro-bial infections One of the main consequences of the biofilmmode of growth is the increased resistance to antimicrobialtherapy resulting in recurrent or persistent infections leadingto treatment failure Therefore biofilms are up to 100-foldmore resistant against any antimicrobial treatment modalityas compared to their planktonic counterpart [102] From thispoint of view it is necessary to evaluate additionally the pho-todynamic efficacy against biofilm growing pathogens of anypositive preselected photosensitizer which has demonstrated


PDT-basedcytotoxicity

Cell deathmode

Microbialdark toxicity

Effect of cell wallpermeabilization

Antimicrobialphototoxicity

Effect onbacterial biofilms

Section 52Section 53

Section 51 Section 51

TissuepenetrationSection 34

Darktoxicity

Section 32

IntracelllocalizationSection 33

Uptakerelease

Section 31


Fluorescenceabsorption spectra

Section 21

Singlet oxygen andradical formation

Section 22

Photostabilityphotobleaching

Section 24

Electric chargemolecular weight

Section 25

(1) Physicalphotochemicalcharacterisation

(2b) PS characterisationfor use in antimicrobial PDI

(2a) PS characterisationfor use in antitumor PDT

Figure 6 Flow chart for basic characterization of novel photosensitizers for PDT andor PDI applicationsThis diagram provides a suggestedstepwise procedure for basic and in vitro characterization of novel PS molecules involving the most important physical photochemical andcellular characteristics The respective sections within this paper are provided

photodynamic killing efficacy against microorganisms insuspension

6 Conclusion

Taken together the previously mentioned experimentalapproaches are suited to provide comprehensive informationon the potential use of a particularmdashand probably newlydevelopedmdashPS agent in the frame of PDT or PDI As severalaspects determine the possibilities of use in the two majorbranches of photodynamic applications (see the aforemen-tioned for details) we suggest a stepwise characterization ofthe most important physical and photochemical-dynamicfeatures of a new PS in order to determine the possible fieldsof therapeutic applications Such a step-by-step approach isdepicted in Figure 6 (with reference to the chapters withinthis paper) providing an interdisciplinary straight forwardstrategy for comprehensive characterization of photosensi-tizing compounds Early and unequivocal identification ofthe various strengths and weaknesses of an individual agentmay help deciding for which particular clinical applicationthe particular drug is worth further establishing

In vitro research represents the initial step in the bio-logical characterization of a new PS for its application inPDT or PDI Conclusions drawn from cell culture experi-ments are always difficult to directly transfer to the in vivosituation and may not allow for a very precise predictionof clinical applications of a given substance However theseexperiments may suggest possible targets and provide firstevidence on practicable PDTPDI treatment protocols Up todate no standard strategy for the basic in vitro investigationsof PS existed Therefore this tutorial which is based onthe authorsrsquo experience in PDT and PDI can serve as a

guide for researchers who are involved in preclinical PStesting or plan to contribute to such research efforts Specialaspects of the experimental categorization of a novel PSsuch as for example the possible interference of the PS withfluorochromes employed in cytological assays or the lightsensitivity of the PS have to be considered by an experimenterwhen performing a spectral analysis the determinationof levels of the phototoxic agents (ROS) including singletoxygen drug and light dose finding intracellular localizationor assessment of the mode of cell death predominant ata given PDT protocol and are discussed in this paperAs outlined before PDI research using microorganisms asmodel systems has to take into account the special natureof these pathogens For example the high growth rate ofbacteria and yeast reasons the requirement of assays whichallow for viability tests covering more than three ordersof magnitude which excludes classical colorimetric assaysFurthermore as discussed in the previous chapters thepresence of cell wall composition and the ability to formbiofilms require alternative PS and therapeutic procedures forsuccessful photokilling when compared to cancer cells

Until now localized infections of the skin wounds infec-tions of the oral cavity infections related to periodontitis andendodontitis as well as infection of the middle ear are espe-cially suitable for PDI treatment because they are relativelyaccessible for PS application and illumination [103] OverallPDImight either be substitute standard antimicrobial therapyor act as an additional approach in the future

Concluding we hope that this tutorial will motivateresearchers of all disciplines to get involved in photodynamictherapy and photodynamic inactivation and thereby helpto further expand the convincing benefits of photodynamicprocedures to new fields of applications


Abbreviations

O2

∙minus Superoxide anionABS AbsorbanceATP Adenosine 51015840-triphosphateBrdU 5-bromo-21015840-deoxyuridineCaspase Cysteine-dependentaspartate-

specificproteaseCFU Colony-forming unitcyt-c Cytochrome cDAPI 410158406-diamidino-2-phenylindoleDC Dark control998779Ψ Mitochondrial membrane potentialDMAX 9-[2-(3-carboxy-910-dimethyl)

anthryl]-6-hydroxy-3H-xanthen-3-oneDMPD NN-dimethyl-p-phenylenediamineDMSO Dimethyl sulphoxideDPA 910-diphenylanthraceneDPBF 13-diphenylisobenzofuranEDTA Ethylenediaminetetraacetic acidET Electron transferFACS Fluorescence-activated cell sortingFBCS Fetal calfbovine serumFI Fluorescence intensityFM Fluorescence microscopyHO∙ Hydroxyl radicalsLM Light microscopyLOC Light-only controlMRSA Methicillin-resistant Staphylococcus aureusMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

ORAC Oxygen-radical absorbance capacityPARP Poly(ADP-ribose) polymerasePDI(B) Photodynamic inactivation (of bacteria)PDT Photodynamic therapyPeT Photo-induced electron transferPI Propidium iodidePS PhotosensitizerPSer PhosphatidylserineROS Reactive oxygen speciesSOSG Singlet Oxygen Sensor GreenTMRE Tetramethylrhodamine ethyl esterTMRM Tetramethylrhodamine methyl esterTRAP Radical-trapping antioxidant parameter

methodTUNEL Terminal deoxynucleotidyl

transferase-mediated dUTP nick endlabeling

UTC Untreated controlWST Water-soluble tetrazolium saltXTT 23-bis-(2-methoxy-4-nitro-5-sulfophenyl)-

2H-tetrazolium-5-carboxanilide

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

A Gollmer and T Maisch have contributed equally to thispaper

References

[1] P Agostinis K Berg KA Cengel et al ldquoPhotodynamic therapyof cancer an updaterdquoACancer Journal for Clinicians vol 61 no4 pp 250ndash281 2011

[2] M S Baptista and M Wainwright ldquoPhotodynamic antimicro-bial chemotherapy (PACT) for the treatment of malaria leish-maniasis and trypanosomiasisrdquoBrazilian Journal ofMedical andBiological Research vol 44 no 1 pp 1ndash10 2011

[3] T Maisch ldquoA new strategy to destroy antibiotic resis-tant microorganisms antimicrobial photodynamic treatmentrdquoMini-Reviews in Medicinal Chemistry vol 9 no 8 pp 974ndash9832009

[4] T Maisch S Hackbarth J Regensburger et al ldquoPhotodynamicinactivation of multi-resistant bacteria (PIB)mdasha new approachto treat superficial infections in the 21st centuryrdquo Journal of theGerman Society of Dermatology vol 9 no 5 pp 360ndash367 2011

[5] G Jori C Fabris M Soncin et al ldquoPhotodynamic therapyin the treatment of microbial infections basic principles andperspective applicationsrdquo Lasers in Surgery and Medicine vol38 no 5 pp 468ndash481 2006

[6] G Jori M Magaraggia C Fabris et al ldquoPhotodynamic inac-tivation of microbial pathogens disinfection of water andprevention of water-borne diseasesrdquo Journal of EnvironmentalPathology Toxicology and Oncology vol 30 no 3 pp 261ndash2712011

[7] K Plaetzer B Krammer J Berlanda F Berr and T KiesslichldquoPhotophysics and photochemistry of photodynamic therapyfundamental aspectsrdquo Lasers in Medical Science vol 24 no 2pp 259ndash268 2009

[8] A Juzeniene K P Nielsen and J Moan ldquoBiophysical aspectsof photodynamic therapyrdquo Journal of Environmental PathologyToxicology and Oncology vol 25 no 1-2 pp 7ndash28 2006

[9] V Engelhardt B Krammer and K Plaetzer ldquoAntibacterialphotodynamic therapy using water-soluble formulations ofhypericin or mTHPC is effective in inactivation of Staphylococ-cus aureusrdquo Photochemical and Photobiological Sciences vol 9no 3 pp 365ndash369 2010

[10] A Kubin H G Loew U Burner G Jessner H Kolbabek andFWierrani ldquoHow tomake hypericinwater-solublerdquoPharmazievol 63 no 4 pp 263ndash269 2008

[11] S Bagdonas L W Ma V Iani R Rotomskis P Juzenasand J Moan ldquoPhototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro a spectroscopic studyrdquoPhotochemistry and Photobiology vol 72 no 2 pp 186ndash1922000

[12] J Moan G Streckyte S Bagdonas O Bech and K BergldquoPhotobleaching of protoporphyrin IX in cells incubated with5-aminolevulinic acidrdquo International Journal of Cancer vol 70no 1 pp 90ndash97 1997

[13] P Charlesworth and T G Truscott ldquoThe use of 5-aminolevulinic acid (ALA) in photodynamic therapy (PDT)rdquoJournal of Photochemistry and Photobiology B vol 18 no 1 pp99ndash100 1993

[14] R W Redmond and I E Kochevar ldquoSpatially resolved cellularresponses to singlet oxygenrdquo Photochemistry and Photobiologyvol 82 no 5 pp 1178ndash1186 2006


[15] P R Ogilby ldquoSinglet oxygen there is indeed something newunder the sunrdquoChemical Society Reviews vol 39 no 8 pp 3181ndash3209 2010

[16] HWuQ SongG Ran X Lu andB Xu ldquoRecent developmentsin the detection of singlet oxygen with molecular spectroscopicmethodsrdquo TrAC Trends in Analytical Chemistry vol 30 no 1pp 133ndash141 2011

[17] C S Foote F C Shook and R A Abakerli ldquoChemistry ofsuperoxide ion 4 Singlet oxygen is not a major product ofdismutationrdquo Journal of the American Chemical Society vol 102no 7 pp 2503ndash2504 1980

[18] V Nardello N Azaroual I Cervoise G Vermeersch andJ M Aubry ldquoSynthesis and photooxidation of sodium 13-cyclohexadiene-14-diethanoate a new colorless and water-soluble trap of singlet oxygenrdquo Tetrahedron vol 52 no 6 pp2031ndash2046 1996

[19] M Botsivali and D F Evans ldquoA new trap for singlet oxygenin aqueous solutionrdquo Journal of the Chemical Society ChemicalCommunications no 24 pp 1114ndash1116 1979

[20] B A Lindig M A J Rodgers and A P Schaap ldquoDetermi-nation of the lifetime of singlet oxygen in D2O using 910-anthracenedipropionic acid a water-soluble proberdquo Journal ofthe American Chemical Society vol 102 no 17 pp 5590ndash55931980

[21] J M Aubry J Rigaudy and N K Cuong ldquoA water-solublerubrene derivative synthesis properties and trapping of 1O

2

inaqueous-solutionrdquoPhotochemistry and Photobiology vol 33 no2 pp 149ndash153 1981

[22] F Wilkinson W P Helman and A B Ross ldquoRate constantsfor the decay and reactions of the lowest electronically excitedsinglet-state ofmolecular-oxygen in solution An expanded andrevised compilationrdquo Journal of Physical andChemical ReferenceData vol 24 no 2 pp 663ndash1021 1995

[23] D R Adams and F Wilkinson ldquoLifetime of singlet oxygenin liquid solutionrdquo Journal of the Chemical Society FaradayTransactions vol 68 pp 586ndash593 1972

[24] A A Gorman G Lovering and M A J Rodgers ldquoA pulseradiolysis study of the triplet sensitized production of singletoxygen determination of energy transfer efficienciesrdquo Journalof the AmericanChemical Society vol 100 no 14 pp 4527ndash45321978

[25] P B Merkel and D R Kearns ldquoRadiationless decay of singletmolecular oxygen in solution Experimental and theoreticalstudy of electronic-to-vibrational energy transferrdquo Journal of theAmerican Chemical Society vol 94 no 21 pp 7244ndash7253 1972

[26] R D Scurlock and P R Ogilby ldquoEffect of solvent on therate constant for the radiative deactivation of singlet molecularoxygen (1ΔgO2)rdquoThe Journal of Physical Chemistry vol 91 no17 pp 4599ndash4602 1987

[27] E J Corey and W C Taylor ldquoA study of the peroxidationof organic compounds by externally generated singlet oxygenmoleculesrdquo Journal of the American Chemical Society vol 86no 18 pp 3881ndash3882 1964

[28] H H Wasserman J R Scheffer and J L Cooper ldquoSinglet oxy-gen reactions with 910-diphenylanthracene peroxiderdquo Journalof the American Chemical Society vol 94 no 14 pp 4991ndash49961972

[29] N Umezawa K Tanaka Y Urano K Kikuchi T Higuchiand T Nagano ldquoNovel fluorescent probes for singlet oxygenrdquoAngewandte Chemie vol 38 no 19 pp 2899ndash2901 1999

[30] K Tanaka T Miura N Umezawa et al ldquoRational designof fluorescein-based fluorescence probes Mechanism-based

design of a maximum fluorescence probe for singlet oxygenrdquoJournal of the American Chemical Society vol 123 no 11 pp2530ndash2536 2001

[31] Singlet Oxygen Sensor Green Reagent Product InformationInvitrogenMolecular Probes 2004

[32] X Ragas A Jimenez-Banzo D Sanchez-Garcıa X Batllori andS Nonell ldquoSinglet oxygen photosensitisation by the fluorescentprobe singlet oxygen sensor greenrdquo Chemical Communicationsno 20 pp 2920ndash2922 2009

[33] A Gollmer J Arnbjerg F H Blaikie et al ldquoSinglet oxygensensor green photochemical behavior in solution and in amammalian cellrdquo Photochemistry and Photobiology vol 87 no3 pp 671ndash679 2011

[34] K Nakamura K Ishiyama H Ikai et al ldquoReevaluation ofanalytical methods for photogenerated singlet oxygenrdquo Journalof Clinical Biochemistry and Nutrition vol 49 no 2 pp 87ndash952011

[35] J W Snyder E Skovsen J D C Lambert and P R OgilbyldquoSubcellular time-resolved studies of singlet oxygen in singlecellsrdquo Journal of the American Chemical Society vol 127 no 42pp 14558ndash14559 2005

[36] J W Snyder E Skovsen J D C Lambert L Poulsen and P ROgilby ldquoOptical detection of singlet oxygen from single cellsrdquoPhysical Chemistry Chemical Physics vol 8 no 37 pp 4280ndash4293 2006

[37] S Hatz J D C Lambert and P R Ogilby ldquoMeasuring thelifetime of singlet oxygen in a single cell addressing the issue ofcell viabilityrdquo Photochemical and Photobiological Sciences vol 6no 10 pp 1106ndash1116 2007

[38] E Skovsen J W Snyder J D C Lambert and P R OgilbyldquoLifetime and diffusion of singlet oxygen in a cellrdquoThe Journalof Physical Chemistry B vol 109 no 18 pp 8570ndash8573 2005

[39] S Hatz L Poulsen and P R Ogilby ldquoTime-resolved singletoxygen phosphorescence measurements from photosensitizedexperiments in single cells effects of oxygen diffusion andoxygen concentrationrdquo Photochemistry and Photobiology vol84 no 5 pp 1284ndash1290 2008

[40] C Schweitzer and R Schmidt ldquoPhysical mechanisms of genera-tion and deactivation of singlet oxygenrdquo Chemical Reviews vol103 no 5 pp 1685ndash1757 2003

[41] L K Andersen and P R Ogilby ldquoTime-resolved detection ofsinglet oxygen in a transmission microscoperdquo Photochemistryand Photobiology vol 73 no 5 pp 489ndash492 2001

[42] J Baier T Maisch M Maier M Landthaler and W BaumlerldquoDirect detection of singlet oxygen generated by UVA irradia-tion in human cells and skinrdquo Journal of Investigative Dermatol-ogy vol 127 no 6 pp 1498ndash1506 2007

[43] M Niedre M S Patterson and B C Wilson ldquoDirect near-infrared luminescence detection of singlet oxygen generatedby photodynamic therapy in cells in vitro and tissues in vivordquoPhotochemistry and Photobiology vol 75 no 4 pp 382ndash3912002

[44] X Ragas X He M Agut et al ldquoSinglet oxygen in antimicrobialphotodynamic therapy photosensitizer-dependent productionand decay in E colirdquo Molecules vol 18 no 3 pp 2712ndash27252013

[45] WM Sharman C M Allen and J E Van Lier ldquoPhotodynamictherapeutics basic principles and clinical applicationsrdquo DrugDiscovery Today vol 4 no 11 pp 507ndash517 1999

[46] M Ochsner ldquoPhotophysical and photobiological processes inthe photodynamic therapy of tumoursrdquo Journal of Photochem-istry and Photobiology B vol 39 no 1 pp 1ndash18 1997


[47] A Felgentrager T Maisch D Dobler and A Spath ldquoHydrogenbond acceptors and additional cationic charges in methyleneblue derivatives photophysics and antimicrobial efficiencyrdquoBioMed Research International vol 2013 Article ID 482167 12pages 2013

[48] J Ferreira P F C Menezes C Kurachi C Sibata R RAllison and V S Bagnato ldquoPhotostability of different chlorinephotosensitizersrdquo Laser Physics Letters vol 5 no 2 pp 156ndash1612008

[49] M B Ericson S Grapengiesser F Gudmundson et al ldquoAspectroscopic study of the photobleaching of protoporphyrin IXin solutionrdquo Lasers in Medical Science vol 18 no 1 pp 56ndash622003

[50] F F C Menezes C A S Melo V S Bagnato H Imasato andJ R Perussi ldquoDark cytotoxicity of the photoproducts of thephotosensitizer photogem after photobleaching induced by aLaserrdquo Computer Modeling in Engineering and Sciences vol 7no 3 pp 435ndash442 2005

[51] J Moan ldquoEffect of bleaching of porphyrin sensitizers duringphotodynamic therapyrdquo Cancer Letters vol 33 no 1 pp 45ndash531986

[52] W G Roberts K M Smith J L McCullough and M WBerns ldquoSkin photosensitivity and photodestruction of severalpotential photodynamic sensitizersrdquo Photochemistry and Pho-tobiology vol 49 no 4 pp 431ndash438 1989

[53] M T Jarvi M S Patterson and B C Wilson ldquoInsights intophotodynamic therapy dosimetry simultaneous singlet oxygenluminescence and photosensitizer photobleaching measure-mentsrdquo Biophysical Society vol 102 no 3 pp 661ndash671 2012

[54] TMaisch JWagner V Papastamou et al ldquoCombination of 10EDTA Photosan and a blue light hand-held photopolymerizerto inactivate leading oral bacteria in dentistry in vitrordquo Journalof Applied Microbiology vol 107 no 5 pp 1569ndash1578 2009

[55] YNitzanMGutterman ZMalik and B Ehrenberg ldquoInactiva-tion of gram-negative bacteria by photosensitized porphyrinsrdquoPhotochemistry and Photobiology vol 55 no 1 pp 89ndash96 1992

[56] F Yoshimura andHNikaido ldquoDiffusion of120573-lactam antibioticsthrough the porin channels of Escherichia coli K-12rdquoAntimicro-bial Agents and Chemotherapy vol 27 no 1 pp 84ndash92 1985

[57] T N Demidova and M R Hamblin ldquoPhotodynamic therapytargeted to pathogensrdquo International Journal of Immunopathol-ogy and Pharmacology vol 17 no 3 pp 245ndash254 2004

[58] A T Michael-Titus R Whelpton and Z Yaqub ldquoBindingof temoporfin to the lipoprotein fractions of human serumrdquoBritish Journal of Clinical Pharmacology vol 40 no 6 pp 594ndash597 1995

[59] H J Hopkinson D I Vernon and S B Brown ldquoIdentificationand partial characterization of an unusual distribution of thephotosensitizer meta-tetrahydroxyphenyl chlorin (temoporfin)in human plasmardquo Photochemistry and Photobiology vol 69 no4 pp 482ndash488 1999

[60] V Engelhardt T Kiesslich J Berlanda S Hofbauer B Kram-mer and K Plaetzer ldquoLipophilic rather than hydrophilic pho-tosensitizers show strong adherence to standard cell culturemicroplates under cell-free conditionsrdquo Journal of Photochem-istry and Photobiology B vol 103 no 3 pp 222ndash229 2011

[61] V Ziegler T Kiesslich B Krammer andK Plaetzer ldquoPhotosen-sitizer adhered to cell culture microplates induces phototoxicityin carcinoma cellsrdquo BioMed Research International vol 2012Article ID 549498 11 pages 2012

[62] R R Allison and C H Sibata ldquoOncologic photodynamictherapy photosensitizers a clinical reviewrdquo Photodiagnosis andPhotodynamic Therapy vol 7 no 2 pp 61ndash75 2010

[63] W Meyer R Schwarz and K Neurand ldquoThe skin of domesticmammals as a model for the human skin with special referenceto the domestic pigrdquoCurrent problems in dermatology vol 7 pp39ndash52 1978

[64] A Eichner F P Gonzales A Felgentrager et al ldquoDirty handsphotodynamic killing of human pathogens like EHEC MRSAand Candida within secondsrdquo Photochemical and Photobiologi-cal Sciences vol 12 no 1 pp 135ndash147 2012

[65] J D Bos and M M H M Meinardi ldquoThe 500 Dalton rulefor the skin penetration of chemical compounds and drugsrdquoExperimental Dermatology vol 9 no 3 pp 165ndash169 2000

[66] A C Williams and B W Barry ldquoPenetration enhancersrdquoAdvanced Drug Delivery Reviews vol 56 no 5 pp 603ndash6182004

[67] J Berlanda T Kiesslich C B Oberdanner F J ObermairB Krammer and K Plaetzer ldquoCharacterization of apoptosisinduced by photodynamic treatment with hypericin in A431human epidermoid carcinoma cellsrdquo Journal of EnvironmentalPathology Toxicology and Oncology vol 25 no 1-2 pp 173ndash1882006

[68] G E N Kass J E Eriksson M Weis S Orrenius and SC Chow ldquoChromatin condensation during apoptosis requiresATPrdquo Biochemical Journal vol 318 no 3 pp 749ndash752 1996

[69] H T Liu R Z Sabirov and Y Okada ldquoOxygen-glucosedeprivation induces ATP release via maxi-anion channels inastrocytesrdquo Purinergic Signalling vol 4 no 2 pp 147ndash154 2008

[70] K Plaetzer T Kiesslich B Krammer and PHammerl ldquoCharac-terization of the cell death modes and the associated changes incellular energy supply in response to AlPcS4-PDTrdquo Photochem-ical and Photobiological Sciences vol 1 no 3 pp 172ndash177 2002

[71] N Yasuhara Y Eguchi T Tachibana N Imamoto Y Yonedaand Y Tsujimoto ldquoEssential role of active nuclear transport inapoptosisrdquo Genes to Cells vol 2 no 1 pp 55ndash64 1997

[72] H Zou Y Li X Liu and X Wang ldquoAn APAF-1cytochrome cmultimeric complex is a functional apoptosome that activatesprocaspase-9rdquoThe Journal of Biological Chemistry vol 274 no17 pp 11549ndash11556 1999

[73] K Plaetzer T Kiesslich C B Oberdanner and B KrammerldquoApoptosis following photodynamic tumor therapy inductionmechanisms and detectionrdquo Current Pharmaceutical Designvol 11 no 9 pp 1151ndash1165 2005

[74] M Oertel S I Schastak A Tannapfel et al ldquoNovel bacteri-ochlorine for high tissue-penetration photodynamic propertiesin human biliary tract cancer cells in vitro and inmouse tumourmodelrdquo Journal of Photochemistry and Photobiology B vol 71no 1-3 pp 1ndash10 2003

[75] J Berlanda T Kiesslich V Engelhardt B Krammer and KPlaetzer ldquoComparative in vitro study on the characteristicsof different photosensitizers employed in PDTrdquo Journal ofPhotochemistry and Photobiology B vol 100 no 3 pp 173ndash1802010

[76] T Kiesslich J Berlanda K Plaetzer B Krammer and F BerrldquoComparative characterization of the efficiency and cellularpharmacokinetics of Foscan- and Foslip-based photodynamictreatment in human biliary tract cancer cell linesrdquo Photochemi-cal and Photobiological Sciences vol 6 no 6 pp 619ndash627 2007

[77] W T Li H W Tsao Y Y Chen S W Cheng and Y CHsu ldquoA study on the photodynamic properties of chlorophyll


derivatives using human hepatocellular carcinoma cellsrdquo Pho-tochemical and Photobiological Sciences vol 6 no 12 pp 1341ndash1348 2007

[78] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[79] T Kiesslich N Tortik D Neureiter M Pichler and KPlaetzer ldquoApoptosis in cancer cells induced by photodynamictreatmentmdasha methodological approachrdquo Journal of Porphyrinsand Phthalocyanines vol 17 pp 197ndash209 2013

[80] E M Czekanska ldquoAssessment of cell proliferation withresazurin-based fluorescent dyerdquoMethods inMolecular Biologyvol 740 pp 27ndash32 2011

[81] T Kiesslich C B Oberdanner B Krammer and K PlaetzerldquoFast and reliable determination of intracellular ATP from cellscultured in 96-well microplatesrdquo Journal of Biochemical andBiophysical Methods vol 57 no 3 pp 247ndash251 2003

[82] D Bach J FuerederM Karbiener et al ldquoComprehensive analy-sis of alterations in the miRNome in response to photodynamictreatmentrdquo Journal of Photochemistry and Photobiology B vol120 pp 74ndash81 2013

[83] Y Luo and D Kessel ldquoInitiation of apoptosis versus necrosis byphotodynamic therapy with chloroaluminum phthalocyaninerdquoPhotochemistry and Photobiology vol 66 no 4 pp 479ndash4831997

[84] C B Oberdanner T Kiesslich B Krammer and K PlaetzerldquoGlucose is required tomaintain highATP-levels for the energy-utilizing steps during PDT-induced apoptosisrdquo Photochemistryand Photobiology vol 76 no 6 pp 695ndash703 2002

[85] S W Perry J P Norman J Barbieri E B Brown and H AGelbard ldquoMitochondrial membrane potential probes and theproton gradient a practical usage guiderdquo Biotechniques vol 50no 2 pp 98ndash115 2011

[86] I Mickuviene V Kirveliene and B Juodka ldquoExperimentalsurvey of non-clonogenic viability assays for adherent cells invitrordquo Toxicology in Vitro vol 18 no 5 pp 639ndash648 2004

[87] D Grebenova H Cajthamlova J Bartosova et al ldquoSelec-tive destruction of leukaemic cells by photo-activation of 5-aminolaevulinic acid-induced protoporphyrin-IXrdquo Journal ofPhotochemistry andPhotobiology B vol 47 no 1 pp 74ndash81 1998

[88] J Wang T Stachon T Eppig A Langenbucher B Seitz and NSzentmary ldquoImpact of photodynamic inactivation (PDI) usingthe photosensitizer chlorin e6 on viabilityrdquo Graefersquos Archive forClinical and Experimental Ophthalmology vol 251 no 4 pp1199ndash1204 2013

[89] D B Zorov E KobrinskyM Juhaszova and S J Sollott ldquoExam-ining intracellular organelle function using fluorescent probesfrom animalcules to quantum dotsrdquo Circulation Research vol95 no 3 pp 239ndash252 2004

[90] GMakin and J AHickman ldquoApoptosis and cancer chemother-apyrdquo Cell and Tissue Research vol 301 no 1 pp 143ndash152 2000

[91] D J McConkey ldquoBiochemical determinants of apoptosis andnecrosisrdquo Toxicology Letters vol 99 no 3 pp 157ndash168 1998

[92] C M Brackett and S O Gollnick ldquoPhotodynamic therapyenhancement of anti-tumor immunityrdquo Photochemical andPhotobiological Sciences vol 10 no 5 pp 649ndash652 2011

[93] F H van Duijnhoven R I J M Aalbers J P Rovers OT Terpstra and P J K Kuppen ldquoThe immunological con-sequences of photodynamic treatment of cancer a literaturereviewrdquo Immunobiology vol 207 no 2 pp 105ndash113 2003

[94] D Kessel and N L Oleinick ldquoInitiation of autophagy byphotodynamic therapyrdquo Methods in Enzymology vol 453 noC pp 1ndash16 2009

[95] J J Reiners P Agostinis K Berg N L Oleinick and D KesselldquoAssessing autophagy in the context of photodynamic therapyrdquoAutophagy vol 6 no 1 pp 7ndash18 2010

[96] D V Krysko T Vanden Berghe K DrsquoHerde and P Vanden-abeele ldquoApoptosis and necrosis detection discrimination andphagocytosisrdquoMethods vol 44 no 3 pp 205ndash221 2008

[97] R Sgonc and J Gruber ldquoApoptosis detection an overviewrdquoExperimental Gerontology vol 33 no 6 pp 525ndash533 1998

[98] D T Loo ldquoIn situ detection of apoptosis by the TUNEL assayan overview of techniquesrdquo Methods in Molecular Biology vol682 pp 3ndash13 2011

[99] C Stadelmann and H Lassmann ldquoDetection of apoptosis intissue sectionsrdquo Cell and Tissue Research vol 301 no 1 pp 19ndash31 2000

[100] J M Boyce and D Pittet ldquoGuideline for hand hygienein health-care settings recommendations of the health-care infection control practices advisory committee and theHIPACSHEAAPICIDSA hand hygiene task forcerdquo AmericanJournal of Infection Control vol 30 no 8 pp S1ndashS46 2002

[101] M Hulsmann M Heckendorff and A Lennon ldquoChelatingagents in root canal treatment mode of action and indicationsfor their userdquo International Endodontic Journal vol 36 no 12pp 810ndash830 2003

[102] J Chandra P K Mukherjee S D Leidich et al ldquoAntifungalresistance of candidal biofilms formed on denture acrylic invitrordquo Journal of Dental Research vol 80 no 3 pp 903ndash9082001

[103] TDai Y YHuang andMRHamblin ldquoPhotodynamic therapyfor localized infections-state of the artrdquo Photodiagnosis andPhotodynamic Therapy vol 6 no 3-4 pp 170ndash188 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and parasites The lack of new antibiotics classes combinedwith the propagation of multidrug resistant bacteriafungiand the economic and regulatory challenges thereof haveboosted the research on PDI [2ndash4] As the properties ofideal PS for PDT and PDI differ numerous new substancesespecially synthesized for this promising approach are beingdeveloped in the photodynamic research community andexpand the field of applications (eg to water-borne diseases)[5 6]

Photodynamic therapy and PDI require as multidisci-plinary approaches the tight cooperation of chemists (egfor synthesis of new PS) biologists (for testing new sub-stances) physicists (eg for light dosimetry) and clinicians(for the transfer from the lab bench to the clinical applica-tion) The development of novel PDTPDI applications inbiomedicine and biotechnology is mainly driven by chemistsand biologists The former design new PS with promisingproperties sometimes without the claim to intend the newsubstance for a specific target and the latter test these newdyes in vitro on eukaryotic or prokaryotic cell cultures inorder to estimate the possible field of application of a PSAlthough many experimental steps of PS testing make useof the wide spectrum of methods readily employed in cellbiology some special aspects (eg fluorescence of the PSmolecule or the requirement of subdued light conditions)have need of being considered when doing in vitro experi-ments in PDTPDI Up to date the scientific photodynamiccommunity did neither suggest a standard strategy for PStesting nor does a tutorial on PS testing exist

The aimof this paper is therefore to provide a comprehen-sive overview of experimental strategies and methodsmdashwithout extensive referencing in order to maintain readabil-itymdashby which novel photoactive drugs can be tested invitro for their employment in photodynamic proceduresIt shall represent a suggestion of operative instructions bywhich novel photoactive molecules may be identified assuitable for PDT and PDI based on photochemical andphotophysical properties of a PS its uptake into cells theintracellular localization and photodynamic action in bothtumor cells and microorganismsThis tutorial shall stimulatethe efforts to expand the convincing benefits of photody-namic procedures within both established and new fields ofapplications and motivate (young) scientists to contribute tothe photodynamic research

2 Characterization of the Photochemical andPhotophysical Properties

21 Recording of AbsorptionFluorescence Spectra in VariousSolventsCells The absorptionexcitation wavelength(s) of agiven PS represents a key selection criterion for its applicationin PDT or PDI As a common agreement UV activation ofphotoactive drugs is inadmissible to exclude damage set tothe cellsrsquo genetic information Also light wavelengths whichare absorbed by themajor tissue or cell chromophores shouldbe avoided for excitation of the PS For PDT on solid tumorsthe effective penetration depth of the excitation light highlydepends on the interference with the absorption spectra

of the major tissue chromophores namely (oxy-deoxy-)hemoglobin and melanin as well as water The absorptionspectra of these molecules define the optical window forPDT in tissue which covers the wavelength range of 600ndash850 nm [7] The upper limit of this window is set by theminimal quantum energy which is required for an efficientproduction of singlet oxygen considering thermal loss ofenergy combined with the shifts of the electrons during thephotophysical processes [8] Due to a different compositionof chromophores in microorganisms the lower wavelengthlimit of the optical window defined for PDT applicationsdoes not necessarily apply for photosensitizers employed inPDI

As the photophysical processes in a fluorescing moleculeare dependent on the solvent excitation and emission spectraof a PS should be read in aqueous solutions (buffers) orbiocompatible solvents such as dimethyl sulphoxide (DMSO)or ethanol In order to increase the water solubility ofrather lipophilic PS fetal calfbovine serum (FCBS) or othersolubility enhancers such as polyvinylpyrrolidone [9 10] maybe added for recording of the spectra In very rare cases(eg for photosensitizers showing absorption peaks with anarrow spectral half-width in combination with laser lightillumination) the recording of spectra of the photosensitizerinside the cells might be necessary to assure a wavelengthoverlap of the light used for excitation with the absorptionof the dye Here cells are incubated with the photosensitizerfor a sufficient period of time detached from the surfaceof the cell culture receptacle (eg by trypsinization) andwashed with buffer to remove PS not internalized into cellsFluorescence spectrometers allowing for stirring the solutionof cells inside the cuvettes might be necessary and the spectrahave to be corrected by samples with cells and without PS[11 12]

Additionally the experimenter has to take into accountthat not only the primary PS itself but also secondarymolecules with different absorption resulting from photo-modification of the primary PS may contribute to the overallphotodynamic efficiency [11 13]

The preparation of stock solutions of the photosensitizingagents might prove useful as they might depending onthe chemical stability of the photosensitizer and the stor-age conditions allow for a good experiment-to-experimentreproducibility of the PS concentration in PDT experimentsIf stock solutions are prepared the final concentration ofsolvents other than physiological buffers in the in vitroapplication should be as low as possible (for DMSO andethanol lt1 vv) and the possible cytotoxic effect of thesolvent should be tested by means of a viability assay (seethe following chapter) under conditions identical to thoseused for incubation of the PS with the target cells Storage ofthe PS should be performed according to the manufacturerrsquosrecommendations or may require individual optimizationThe stock solutions of somePSmay be kept frozen (exceptioneg liposomal formulations) Independent of the storageconditions (as solid powder or in solution) the controlmeasurements of the PS spectral properties on a regularbasis are highly recommended to rule out PS decomposi-tion



2


2




2


2


2


2


2



2


2


2


2


2


2





2



2


2


2



2


2


6 Φph (1O2



2


2


2


O2


2



2



Abso

rptio

n

1270

nm

O OHO

ClCOOH

COOH

Nonfluorescent

O HO

ClCOOH

COOH

OO

Highlyfluorescent

ISC


Detection

PS fl

uore

scen

ce

Lum

ines

cenc

e

1198781

1198791

O

119878119899

1198780 3O2

1O2



HO

ClCOOH

COOH

OO

OHO

ClCOOH

COOH





O O O

1O2



2



2






2

withsubstrates

The 1O2


[1O2

] (119905) =

[1198791

]0

1198961198791Δ

[3O2

]

119896119879

minus 119896119889

(119890minus119896119889119905

minus 119890minus119896119879119905

) (1)

where [1198791

]0


1

state 1198961198791998779


1

state by 1O2

[3O2


119879


1



2


2


2




The 1O2


ΦPh = ΦΔ119896119903120591Δ (2)

where ΦΔ




rarr3O2

and120591Δ

the lifetime of 1O2




2


2





2


2

rarr3O2



















Metabolicenzyme(s)





Apoptotic changes








microscope Yes [67]





ΔΨ




















50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2


2




2


2


2


2


2



2


2


2


2


2


2





2



2


2


2



2


2


6 Φph (1O2



2


2


2


O2


2



2



Abso

rptio

n

1270

nm

O OHO

ClCOOH

COOH

Nonfluorescent

O HO

ClCOOH

COOH

OO

Highlyfluorescent

ISC


Detection

PS fl

uore

scen

ce

Lum

ines

cenc

e

1198781

1198791

O

119878119899

1198780 3O2

1O2



HO

ClCOOH

COOH

OO

OHO

ClCOOH

COOH





O O O

1O2



2



2






2

withsubstrates

The 1O2


[1O2

] (119905) =

[1198791

]0

1198961198791Δ

[3O2

]

119896119879

minus 119896119889

(119890minus119896119889119905

minus 119890minus119896119879119905

) (1)

where [1198791

]0


1

state 1198961198791998779


1

state by 1O2

[3O2


119879


1



2


2


2




The 1O2


ΦPh = ΦΔ119896119903120591Δ (2)

where ΦΔ




rarr3O2

and120591Δ

the lifetime of 1O2




2


2





2


2

rarr3O2



















Metabolicenzyme(s)





Apoptotic changes








microscope Yes [67]





ΔΨ




















50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abso

rptio

n

1270

nm

O OHO

ClCOOH

COOH

Nonfluorescent

O HO

ClCOOH

COOH

OO

Highlyfluorescent

ISC


Detection

PS fl

uore

scen

ce

Lum

ines

cenc

e

1198781

1198791

O

119878119899

1198780 3O2

1O2



HO

ClCOOH

COOH

OO

OHO

ClCOOH

COOH





O O O

1O2



2



2






2

withsubstrates

The 1O2


[1O2

] (119905) =

[1198791

]0

1198961198791Δ

[3O2

]

119896119879

minus 119896119889

(119890minus119896119889119905

minus 119890minus119896119879119905

) (1)

where [1198791

]0


1

state 1198961198791998779


1

state by 1O2

[3O2


119879


1



2


2


2




The 1O2


ΦPh = ΦΔ119896119903120591Δ (2)

where ΦΔ




rarr3O2

and120591Δ

the lifetime of 1O2




2


2





2


2

rarr3O2



















Metabolicenzyme(s)





Apoptotic changes








microscope Yes [67]





ΔΨ




















50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2


2

rarr3O2



















Metabolicenzyme(s)





Apoptotic changes








microscope Yes [67]





ΔΨ




















50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Metabolicenzyme(s)





Apoptotic changes








microscope Yes [67]





ΔΨ




















50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Metabolicenzyme(s)





Apoptotic changes








microscope Yes [67]





ΔΨ




















50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










50










50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







50


50





50







Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Blan

ksD

CsU

TCs Treated samples

ABCDEFGH

10 11 12




Relat

ive v

iabi

lity

( U

TC)

Relat

ive v

iabi

lity

( U

TC)

100908070605040302010

0

DCs

UTC

s

PS conc 1PS conc 2

Treated samples

100908070605040302010

0

DCs

UTC

s


(i)

(ii)



Nolight

1 2 3 4 5 6 7 8 9





Cel

lula

r res

pons

e









DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




DNA degradation






FM Quantitative


















(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(b)

(c)

1101009080706050403020100

minus10

minus10

Early reading

Late reading

110100

908070605040302010


Difference curve

Early reading

Late reading


Apoptosis Necrosis

(eg 2-3 hrs pi)

(eg 24 hrs pi)

= 1199051

= 1199052

Rela

tive v

iabi

lity

( U

TC)

Rela

tive v

iabi

lity

( U

TC)

Δ(1199051 1199052)

Δ(1199051 1199052) lt 0 Δ(1199051 1199052) gt 0 Δ(1199051 1199052) sim 0





10




10


10







Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Cell deathmode








Darktoxicity

Section 32


Uptakerelease

Section 31



Section 21


Section 22


Section 24


Section 25






6 Conclusion







Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abbreviations

O2














References























2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









2































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article A Comprehensive Tutorial on In Vitro ...downloads.hindawi.com/journals/bmri/2013/840417.pdfPhotodynamic therapy and PDI require, as multidisci-plinary approaches, the