A Comparative Study of the Photosensitizing Characteristics of Some Cyanine Dyes

1011-1344/00/$ - see front matter q2000 Elsevier Science S.A. All rights reserved.PII S 1011- 1344 (00)00021 -X

Wednesday May 03 09:52 AM StyleTag -- Journal: JPB (J. Photochem. Photobiol. B: Biol.) Article: 7933

www.elsevier.nl/locate/jphotobiolJ. Photochem. Photobiol. B: Biol. 55 (2000) 2736

A comparative study of the photosensitizing characteristics ofsome cyanine dyes

E. Delaey a, F. van Laar b, D. De Vos b, A. Kamuhabwa a, P. Jacobs b, P. de Witte a,*a Laboratorium voor Farmaceutische Biologie en Fytofarmacologie, Faculteit Farmaceutische Wetenschappen, K.U. Leuven, B-3000 Leuven, Belgium

b Centrum voor Oppervlaktechemie en Katalyse, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, K.U. Leuven,B-3001 Heverlee, Belgium

Received 21 December 1999; accepted 27 January 2000

Abstract

The present work has been carried out to explore the potential application of cyanines in photodynamic therapy. After photosensitization,the in vitro cytotoxic and antiproliferative activity on HeLa cells of a total of 35 cyanines belonging to several chemical subgroups is explored.Most of these cyanines have never been used before in similar experimental work. From a first set of experiments, it is found that none of thekrypto-, oxa- and imidacyanines is photobiologically active on HeLa cells. Conversely, five thiacyanines (Thiac15), one rhodacyanine(Rhodac) and four indocyanines (Indoc2, Indoc4, Indoc5, Indoc7) show photodependent cytotoxicity or antiproliferative effects. A moredetailed study shows that out of the ten selected compounds, eight cyanines feature significant photodependent cytotoxic and antiproliferativeeffects. All possess maximum absorption ranges between 545 and 824 nm. In particular, Rhodac, a tetramethinemeromonomethine rhodacy-anine dye with an absorption maximum of 655 nm (ethanol) and a molar absorption coefficient s108 000 shows very promising photo-dependent biological activity. In general, the measured singlet oxygen quantum yield of the selected cyanines is low (-0.08) and does notcorrelate with the degree of photosensitization. Furthermore, the present study shows that cyanines with a partition coefficient close to 1.5accumulate to the highest extent in HeLa cells, while the more hydrophobic compounds (e.g., indocyanines) concentrate lessintracellularly. q2000 Elsevier Science S.A. All rights reserved.

Keywords: Carbocyanines; Photosensitization; Cytotoxicity; HeLa; Cellular uptake

1. Introduction

Photodynamic therapy (PDT) is an alternative modalityin cancer treatment and is based on the use of photosensitizingchemicals that preferentially accumulate in target tumor cells.Photofrin, which is a synthetic hematoporphyrin derivative(HPD), is commonly used in clinical trials for PDT of variouscancers. Although this HPD is efficacious and safe in thetreatment of different human cancers, it has several disadvan-tages, such as its complex chemical composition, the longretention time in several types of normal tissue (about 46weeks) and weak absorbance above 600 nm [1]. Therefore,new photosensitizers are being developed with increasedchemical purity, low dark systemic toxicity, strongabsorptionin the phototherapeutic window from 600 to 1000 nm andpreferential tumor localization [2]. So far, no single photo-sensitizer is available that exhibits all of these properties, andthe search for ideal photosensitizing drugs continues.

* Corresponding author. Tel.: q32-16-323-432; fax: q32-16-323-460;e-mail: [email protected]

Cyanines have been studied as potential PDT tools duringthe last decade [3,4]. The economic interest in these dyesgoes back to their effectiveness as photographic sensitizersand numerous compounds are commercially available. Cya-nines consist of two heterocycles linked by a monomethinebridge, while carbocyanines and dicarbocyanines contain anoligomethine bridge (Figs. 1 and 2). Resonance between thering systems creates chromophores that absorb in the visibleregion, frequently with high molar absorption coefficients.Most of these compounds are cationic, in contrast to the morefrequently used anionic photosensitizers such as hematopor-phyrin derivatives, chlorins and sulfonated phthalocyanines.Since typically an electrical potential gradient of about y180mV exists across the mitochondrial membrane, cationic cya-nines strongly concentrate into mitochondria, up to 1000-foldwith respect to the extracellular concentration [3]. Morespecifically, it has been found that cationic dyes such asEDKC (N,N9-bis(2-ethyl-1,3-dioxolane)kryptocyanine) areretained in vitro and in vivo to a much greater extent in themitochondria of carcinoma and melanoma cells than in nor-

E. Delaey et al. / J. Photochem. Photobiol. B: Biol. 55 (2000) 273628


Fig. 1. The chemical structure of Rhodac (a), Indoc2 (b) and Indoc4 (c).

Fig. 2. The chemical structure of Thiac1-5, Indoc5 and Indoc7.

mal cells [5]. Thus, cationic dyes can be used as tumor-cell-specific photosensitizers with reduced skin phototoxicity anddamage to normal tissue.

However, cyanines featuring a net anionic charge due tothe presence of sulfonate substituents have also been used asselective and effective photosensitizers. For instance, mero-cyanine 540 (MC540), a negatively charged oxacyaninederivative, is used for the selective purging of ocular leuke-mia, lymphoma and neuroblastoma cells in autologous bonemarrow grafts. It is likely that the compound predominantlyfunctions as a membrane-bound 1O2 photogenerator [4,6].More recently indocyanine green [7,8], an anionic indotri-carbocyanine derivative, was also introduced into photome-dicinal practice [7,8].

In this study, we have analyzed the photodynamic actionof 35 cyanines, belonging to several chemical subgroups, onHeLa cells. Furthermore, the in vitro photocytotoxic and anti-proliferative effect of the photoactive cyanines was studiedin more detail. In addition, the cellular accumulation in HeLacells and the partition coefficient of the selected cyanineswere investigated in order to elucidate the background of theobserved dark and photodependent effects. Finally, ESR

E. Delaey et al. / J. Photochem. Photobiol. B: Biol. 55 (2000) 2736 29


Table 1Product name or product number of oxacyanines, thiacyanines, rhodacy-anine, indocyanines, imidacyanines and kryptocyanine and derivatives(available from Aldrich and SigmaAldrich Library of rare chemicals struc-ture index) used in the present study to screen for photocytotoxic com-pounds. After selection, the marked substances (U) were studied in moredetail. The products are preceded by the code names that are used in thispaper for convenience.

OxacyaninesOxac1 3,39-diethyloxacarbocyanine iodideOxac2 3,39-diethyloxadicarbocyanine iodideOxac3 3,39-diethyloxatricarbocyanine iodideOxac4 ethyl-(ethyl-(ethyl-phenyl-benzooxazol-yl)-

buta-dienyl)-phenyl-benzooxazol-3-ium ethanesulfonate

ThiacyaninesThiac1 3,39-diethylthiacarbocyanine iodide (U)Thiac2 3,39-diethyl-9-methylthiacarbocyanine iodide (U)Thiac3 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-

ylidene)-2-methylpropenyl]-naphtho[1,2-d]-thiazolium bromide (U)

Thiac4 3,39-diethylthiadicarbocyanine iodide (U)Thiac5 3,39-diethylthiatricarbocyanine iodide (U)Thiac6 (hydroxy-ethyl)-(((hydroxy-ethyl)-benzothiazol-

ylidene)-methyl-propenyl)-benzothiazolium chlorideThiac7 RCL S17,346-0Thiac8 RCL S17,348-7RhodacyanineRhodac 5-[3-ethoxy-4-(3-ethyl-5-methyl-2(3H)-

benzothiazolyl-idene)-2-butenylidene]-3-ethyl-2-[(3-ethyl-4,5-di-phenyl-2(3H)-thiazolylidene)methyl]-4,5-dihydro-4-oxothiazolium iodide (U)

IndocyaninesIndoc1 indocyanine greenIndoc2 new indocyanine green (U)Indoc3 1,19,3,3,39,39-hexamethyl-indodicarbocyanine iodideIndoc4 1,19,3,3,39,39-hexamethyl-indotricarbocyanine iodide

(U)Indoc5 IR-768 perchlorate (U)Indoc6 IR-780 iodideIndoc7 IR-792 perchlorate (U)Indoc8 IR-1048ImidacyaninesImidac1 RCL S13,111-3Imidac2 RCL S17,120-4Imidac3 RCL S17,123-9Imidac4 RCL S17,132-8Imidac5 direct red 39Imidac6 5-cyano-2-[3-(5-cyano-1,3-diethyl-1,3-dihydro-2H-

benzimidazol-2-ylidene)-1-propenyl]-1-ethyl-3-(4-sulfobutyl)-1H-benzimidazolium hydroxide, inner salt

KryptocyaninesKryptoc1 1,19-diethyl-2,29-cyanine iodideKryptoc2 1,19-diethyl-2,49-cyanine iodideKryptoc3 1,19-diethyl-4,49-cyanine iodideKryptoc4 1,19-diethyl-2,29-carbocyanine iodideKryptoc5 1,19-diethyl-4,49-carbocyanine iodideKryptoc6 1,19-diethyl-4,49-dicarbocyanine iodideKryptoc7 1,19-diethyl-2,29-dicarbocyanine iodideKryptoc8 1,19-diethyl-2,29-quinotricarbocyanine iodide

studies were performed to correlate the photocytotoxic activ-ity of the cyanines with their singlet oxygen quantum yield.

2. Materials and methods

2.1. Compounds and cell culture

Cyanine dyes (Table 1) were obtained from Aldrich (Mil-waukee, WI, USA) and used as received. Hypericin wasprepared as described earlier [9,10]. The compounds weredissolved in dimethylsulfoxide (DMSO) for the cell-cultureexperiments and stored at y208C in dark conditions. Underthese conditions, the solutions were stable for more than twomonths. Photofrin (Ispen Pharma, Ettlingen, Germany) wasreconstituted in 5% dextrose in water at a concentration of25 mg/ml just before use in accordance with the manufac-turers instructions. HeLa cells (cervix carcinoma, human)were obtained from American Type Culture Collection(Rockville, MD, USA). The cells (passage range between10 and 30) were grown at 378C in a humidified 5% CO2 and95% air atmosphere in Minimum Essential Medium (MEM)with Earles salts containing 2 mM L-glutamine, non-essen-tial amino acids (100=), penicillin (100 IU/ml), strepto-mycin (100 mg/ml), tylocine (60 mg/ml), amphotericin B(0.25 mg/ml) and 10% fetal bovine serum (FBS). All cul-ture-medium compounds were purchased from Gibco BRL(Paisley, Scotland). Dilutions of stock solutions were madein cell-culture medium, with a final DMSO concentration of0.1%. This concentration did not affect the cell viability.

2.2. Light irradiation and spectrophotometry

Ninety-six-well plates (Falcon, Franklin Lakes, NJ, USA)were placed 40 cm above a water-cooled 1000 W halogenlamp (Philips) with a prominent spectral output in theabsorption region of the different dyes (Fig. 3(a)). At thesurface of the plates, the uniform fluence rate was 23 mW/cm2, as measured with an IL 1400 radiometer (InternationalLight, Newburyport, MA). The cells were irradiated for 15min. During irradiation, the temperature never exceeded298C. This temperature did not influence the viability of thecells. Visible spectra were recorded in ethanol on an Ultros-pec 2000 UVVis spectrophotometer (Pharmacia, Uppsala,Sweden).

2.3. Cytotoxicity assay

The photocytotoxic effect 1 h after irradiation was deter-mined by assessing cell viability using Neutral Red (Acros,Geel, Belgium), a known specific marker for lysosomalintegrity [11]. HeLa cells were seeded onto 96-well tissue-culture plates at 3=104 cells per well, and were incubatedfor 24 h at 378C. The medium was replaced under strictlysubdued light conditions (-1 mW/cm2), with fresh mediumcontaining different concentrations of dye or DMSO. Sub-

sequently, the cells were incubated under dark conditions at378C for 24 h. Under these conditions, all the cyanines werestable. The drug-containing medium was then replaced with



Fig. 3. The spectral output of the 1000 W halogen lamp (Philips) according to the specifications of the manufacturer (a) and absorption spectra of the differentcompounds (b). Spectra in (b) were normalized at their highest peak.

drug-free medium under subdued light conditions after wash-ing with phosphate-buffered saline (PBS), and cells wereimmediately light irradiated (or not, in the case of darkcytotoxicity). Afterwards cells were incubated at 378C underdark conditions for 45 min. The amount of Neutral Red accu-mulated in the viable cells was measured at 550 nm using amicrotiter plate reader (SLT, Salzburg, Austria) andexpressed as the percentage of dye extracted from untreatedcontrol cells. After curve fitting using non-linear regression(Prism, San Diego, CA, USA), CC50 values (the concentra-tion giving 50% cytotoxicity in comparison with the control)were determined for each experiment. The mean CC50 valuewas calculated from three independent experiments.

2.4. Antiproliferative assay

The antiproliferative assay using HeLa cells was per-formed as reported in Ref. [10]. Briefly, 1=103 cells perwell were seeded onto 96-well plates, and incubated for 24 hat 378C. Subsequently, the cells were washed with PBS andincubated with dye or DMSO for 24 h at 378C, and irradiated

or not. After the irradiation, cells were incubated under darkconditions for three days. Cell proliferation was determinedby quantification of the cellular protein content using Naph-thol Blue Black (Acros, Geel, Belgium), according to themethod of Palombella and Vilcek [12]. The amount of dyewas measured at 620 nm using a microtiter plate reader. Aftercurve fitting using non-linear regression (Prism), the IC50values (the concentration resulting in 50% inhibition of cellproliferation when compared with the control) were deter-mined for each experiment (ns3).

2.5. Cellular accumulation

HeLa cells (9=105) were plated and incubated for 48 hin six-well tissue culture plates (Falcon). They were thenexposed to 1 or 5 mM dye (or DMSO) for 24 h under darkconditions. Subsequently, the cells were rinsed under sub-dued light conditions twice with PBS containing 2% bovineserum albumin (BSA, Fluka, Buchs, Switzerland) and twicewith PBS. Subsequently, 1 ml trypsin (Gibco BRL, Paisley,Scotland) was added to the cells and they were scraped from



the bottom of the culture plate with a rubber policeman. Thenumber of cells recovered per well was determined using acell counter (Coulter Z1 Particle Counter, CoulterElectronic,UK) in all experiments. The cell suspension was then centri-fuged (6000g, 10 min) and the dyes were extracted from thecell pellet with 500 ml DMSO under sonication for 2 min.After centrifugation (6000g, 10 min), the content of Thiac14 and Rhodac present in DMSO was analysed with a micro-plate fluorescence reader (FL600, Bio-tek, Winooski, VT,USA) using calibration curves. The excitation and emissionwavelengths were set at 590 and 645 nm for Thiac3, Thiac4and Rhodac and at 530 and 590 nm for Thiac12. The quan-tification of the other dyes in DMSO was performed by meas-uring the absorbance of the supernatants containing the dyesat lmax. Dye concentrations were calculated by a calibrationcurve. The experiment was done in triplicate.

2.6. Determination of partition coefficients

n-Octanol was purified by shaking with 1N sodiumhydroxide, followed by washing with water until neutral.Partition coefficients were determined by adding 20 mM ofeach compound to 5 ml octanol and an equal volume of PBS(pH 7.4) was added. Each phase was presaturated with theother. The tubes were then vortexed for 1 min at high speedand placed in a shaker for 4 h at room temperature (21228C), followed by centrifugation for 10 min at approxi-mately 500g to separate the octanol and water phase. Thephase containing the larger quantity of the compound beingpartitioned (octanol) was then removed and added to a freshaliquot of the opposite phase, and reprocessed as describedabove. This procedure was repeated twice in order to stabilizethe partition coefficient as described in Ref. [13]. The drugconcentrations in the octanol and in the aqueous phase weremeasured from UV absorbance and deduced from the stan-dard curve. The whole procedure was performed in subduedlight conditions.

2.7. ESR

Photosensitized singlet molecular oxygen (1O2) yieldswere determined by trapping of 1O2 with 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), and measuring the concentra-tion of the formed 2,2,6,6-tetramethyl-4-piperidone-N-oxylradical (4-oxo-TEMPO) with ESR [14,15]. X-band ESRspectra (microwave power, 20 mW; modulation amplitude,3.16 G; 100 kHz field modulation) were recorded with aBruker ESP-300 apparatus at room temperature. Samples(150 ml) were injected into a flat ESR quartz cell and illu-minated directly inside the microwave cavity using a SchottKL-1500 cold light source. 4-oxo-TEMP was purchased fromAcros (Geel, Belgium) and used as received. Typically thereaction mixtures consist of an oxygen-saturated ethanolsolution containing the sensitizer (30 mM) and 4-oxo-TEMP(0.1 M). Spin trapping of 1O2 by 4-oxo-TEMP was per-formed according to a modification of the method of Lion et

al. [16]. 1O2 quantum yields were calculated using RoseBengal as a reference (Fss0.76). The method was con-trolled by measuring the 4-oxo-TEMPO production rateand calculating the 1O2 quantum yields for hypericin(0.73"0.03) and Methylene Blue (0.25"0.03), whichwere in correspondence with literature values. When the 1O2formation was too weak to be detected with the 4-oxo-TEMPreaction, chemical trapping experiments were set up with1-methyl-1-cyclohexene (Acros) (2 mmol 1-methyl-1-cyclo-hexene in 2 ml ethanol, 15 mM sensitizer, 24 h irradiation)[17].

2.8. Statistical analysis

The significance of differences was calculated by usingStudents t-test. Values of p-0.05 were considered to besignificant.

3. Results

3.1. Photodependent cytotoxic and antiproliferative effects

In a preliminary set of experiments, the photodependentand dark cytotoxic and antiproliferative effects of four oxa-cyanines, eight thiacyanines, one rhodacyanine, eight indo-cyanines, six imidacyanines, one kryptocyanine and sevenderivatives (Table 1) were tested on HeLa cells. Hypericinand Photofrin were included as standard photosensitizers. Allcyanine compounds feature a positive charge, except for Imi-dac1, Imidac3 and Imidac6, which are neutral, and Indoc1,Indoc2 and Imidac2, which are anionic compounds due tothe presence of one or two sulfonate groups, respectively(Table 1). Since all dyes showed a large variety of absorptionspectra in the visible spectrum (see below), a lamp with abroad and continuous spectrum was chosen. The spectraloutput of the lamp is shown in Fig. 3(a). HeLa cells wereincubated with 1 and 10 mM of each of the compounds andirradiated (or not) with light after 24 h. The photocytotoxiceffect 1 h after irradiation was determined by assessing cellviability using Neutral Red. Photocytotoxicity tested in thisway therefore assesses early photodependent damage to sub-cellular organelles. Alternatively, the long-term cellulareffect of the photoactivated compounds (1 mM) was testedin an antiproliferation assay. Compounds were considered toexhibit photobiological activity only when the cytotoxicity(at the 1 or 10 mM level) or antiproliferative effect (at the 1mM level) increased by at least an additional 20% after lightirradition, as compared with the results obtained in darkconditions.

Using these criteria, it was found that none of the krypto-,oxa- or imidacyanines was photobiologically active on HeLacells. Conversely, five cationic thiacyanines (Thiac1-5), onecationic rhodacyanine (Rhodac), one anionic (Indoc2) andthree cationic (Indoc4, Indoc5, Indoc7) indocyanines (Figs.1 and 2) showed photodependent cytotoxicity or antiproli-



Table 2The maximum absorption wavelength (lmax), molar absorption coefficient(), singlet oxygen quantum yield (Fs) and logarithm of the octanol/waterpartition coefficient (LogPo/w) for the photosensitizers used.

Cyanine lmax (nm) (My1 cmy1) Fs a LogPo/w

Thiac1 560 144000 0.005"0.001 1.85Thiac2 545 86000 -0.002 2.11Thiac3 575 80000 -0.002 1.82Thiac4 655 232000 0.033 1.94Thiac5 764 199000 0.013 2.10Rhodac 655 108000 0.005"0.001 1.74Indoc2 824 1466000 0.077 n.d. bIndoc4 742 231000 0.054 2.39Indoc5 771 276000 0.027 2.22Indoc7 797 264000 0.011 2.21Hypericin 598 39500 0.73"0.03 n.d. bPhotofrin 630 3200 0.89 c n.d. b

a The means of different experiments are given. R.S.D.-10%, unless statedotherwise.b Not determined.c Literature [35].

Table 3The cytotoxic and antiproliferative effects of the photosensitizers used on HeLa cells determined in dark conditions or after light activation

CC50 (mM) a Fc b IC50 (mM) a FI c Fluence d

light dark light dark (J/cm2)

Thiac1 0.41"0.2 9.1"0.2UUU 22 0.051"0.01 0.19"0.04UU 3.7 3Thiac2 2.1"1 6.2"2U 3.0 0.066"0.03 0.20"0.1 3.0 3Thiac3 0.5"0.04 1.1"0.3U 2.2 0.032"0.009 0.097"0.01UU 3.0 4Thiac4 0.64"0.3 3.5"0.5UU 5.5 0.059"0.02 0.17"0.08 2.9 5Thiac5 4.5"0.8 6.5"2.2 1.4 0.45"0.05 0.80"0.2U 1.8 10Rhodac 0.39"0.1 4.2"1.3UU 11 0.055"0.02 0.63"0.16UU 11 6Indoc2 2.1"0.5 3.1"1 1.5 2.0"0.9 2.2"1.0 1.1 12Indoc4 )10 )10 0.29"0.1 0.69"0.1UU 2.4 9Indoc5 0.60"0.3 2.1"0.5U 3.5 0.061"0.01 0.20"0.07U 3.3 9Indoc7 0.94"0.3 1.0"0.2 1.1 0.29"0.04 0.41"0.09 1.4 11Hypericin 0.83"0.1 )10 )12 0.23"0.08 )10 )43 5Photofrin 5.82"0.27 e )25 e )4.3 2.57"0.12 e )25 e )10 5

a Means"SD of independent experiments (ns3) are given. The mean"SD of the dark conditions was statistically analyzed versus light conditions (Up-0.05,UUp-0.01, UUUp-0.001).b Fc, ratio of the CC50 value (dark) to CC50 value (light).c FI, ratio of the IC50 value (dark) to IC50 value (light).d Fluences were calculated by integrating the spectral output of the lamp over the area defined by the absorption spectrum of each compound (see Fig. 3).e Expressed in mg/ml.

ferative effects. The latter compounds, as well as the standardphotosensitizers hypericin and Photofrin, were studied inmore detail. The absorption spectra of the cyanines and themaximum absorption wavelength as well as the molar absorp-tion coefficients of the photosensitizers used are collected inTable 2 and Fig. 3(b). Since the spectral output of the lampis somewhat variable, the extent of overlap of the absorptionspectrum and the lamp emission was taken into account tounderstand correctly the photosensitizing effect of each ofthe dyes used. For that purpose, the spectral output of thelamp was integrated over the area defined by the differentabsorption peaks (e.g., 600900 nm for Indoc2, see Fig.3(b)) and the corresponding fluences were calculated(Table

3). In the cases of hypericin and Photofrin, which exhibitseveral peaks in the visible spectrum (data not shown), therange 400650 nm was used to calculate the respective flu-ences. From Table 3 it can be seen that the difference betweenthe minimal fluence (3 J/cm2) and the maximal fluence (12J/cm2) used is a factor of four. For each of the selected dyes,the CC50 value (the concentration giving 50% cytotoxicityin comparison with the control) and IC50 value (the concen-tration resulting in 50% inhibition of cell proliferation) deter-mined in dark conditions and after light activation weredetermined. The data show that all of the selected cyanines,except for Indoc2 and Indoc7, exhibited a significant photo-dependent cytotoxic or antiproliferative effect, as indicatedby the ratio of both the CC50 values (Fc) and the IC50 values(FI) (Table 3). No correlation between the calculated flu-ence and the ratio values was found, demonstrating that thelimited photoeffect displayed by a few compounds was notdue to their deficient photoactivation. The results for Thiac1and Thiac4 are consistent with previously reported data [18].As can be noticed further, IC50 values are about one order ofmagnitude lower than the corresponding CC50 values for thecyanines, except for Indoc2, which showed similar IC50 andCC50 values. However, the Fc and FI values obtained in darkand light conditions are approximately the same, except forThiac1. This suggests that for most of the compounds, thedegree of photodamage on top of the effects induced in darkconditions is similar for cytotoxicity and antiproliferativeeffects.

Although structural differences are limited among Thiac14 and Thiac5 (Fig. 2), the least active, marked variationsexist between their photodependent and dark cytotoxic andantiproliferative effects. In the case of Rhodac, a complextetramethinemeromonomethine rhodacyanine dye containing



Fig. 4. The intracellular concentration of the selected cyanines in HeLa cells. The cells were incubated in the dark with 1 mM (open bars) and 5 mM (filledbars) for 24 h. Bars represent the mean"SD of different experiments (ns3).

Fig. 5. The logarithm of the intracellular concentration (logConci) of someselected cyanines in HeLa cells, after a 24 h incubation with 5 mM, as afunction of the logarithm of the partition coefficient (logPO/W). Two com-pounds (Thiac5 and Indoc2) were excluded from this analysis.

a 4-oxothiazolidine ring (rhodanine) bridging two thiazoleheterocycles, the ratio between the dark and light-dependentcytotoxic and antiproliferative effects was as high as 11. Inthis case the calculated fluence was 6 J/cm2, which is closeto the fluence used to activate the standard photosensitizershypericin and Photofrin (5 J/cm2). In general, the data forthe indocyanines are consistent with those for the thiacyani-nes, except for the anionic Indoc2, which displayed an anti-proliferative effect only in the micromolar range. Hypericinand Photofrin, both potent standard photosensitizers, exhib-ited CC50 values of 0.83 mM and 5.8 mg/ml and IC50 valuesof 0.23 mM and 2.57 mg/ml, respectively (Table 3). As canbe seen further, these compounds showed substantially lowercytotoxic and antiproliferative effects in dark conditions thanthe cyanines. These data are in line with previous data ontheir cytotoxic effects after photoactivation [10,1922] andfurther prove the validity of the experimental set-up used inthe present work to screen for photoactive cyanines.

3.2. Cellular accumulation

Cellular accumulation and partition coefficients of theselected cyanines were then investigated to explore furtherthe background of the observed dark and photodependenteffects. For the cellular accumulation experiments, HeLacells were incubated with the selected cyanines (1 and 5 mM)for 24 h. Afterwards the trypsinized cells were counted, andthe dye was extracted and quantified. The final intracellularconcentration was converted to units of mM assuming 3 mlas the mean volume of 106 cells [23]. It can be seen thatThiac3, Thiac4 and Rhodac accumulated dramatically morein HeLa cells than the other compounds (Fig. 4). In the caseof Rhodac, average intracellular concentrations as high as531 ("55) and 1674 ("18) mM were obtained using extra-

cellular concentrations of 1 and 5 mM, respectively. Surpris-ingly, the intracellular concentration of Thiac5, a homologueof Thiac1 and Thiac4, could not be determined due to valueslower than the detection limit (-10 mM). In general, indo-cyanine dyes accumulated in the cells to a somewhat lowerextent. As revealed by the octanol/waterpartitioncoefficients(Table 2, PO/W), all cationic compounds displayed hydro-phobic behavior. It can be seen that cyanines with a relativelylow PO/W were concentrated to a larger extent in the cellsthan those with a higher partition coefficient

.

In general, alinear correlation (R2: 0.68; p-0.01) was found between thelogarithm of the intracellular concentration and the logarithmof PO/W (Fig. 5) when the cells were incubated with 5 mMdye. However, in case of a 1 mM extracellular concentration,the correlation coefficient was somewhat lower (R2: 0.54;p-0.04) (results not shown).



3.3. Singlet oxygen quantum yield

In order to determine the singlet oxygen quantum yieldsof the selected cyanines and hypericin, 4-oxo-TEMP wasreacted with 1O2 to generate the stable radical4-oxo-TEMPO,which can easily be detected with ESR [14,15]. 4-oxo-TEMPO formation is directly related to the 1O2-generatingquantum yields. The singlet oxygen quantum yields of thephotosensitizers used are mentioned in Table 2. For Thiac1,Thiac2, Thiac3 and Rhodac, the formation of 4-oxo-TEMPOcould not be detected with ESR upon irradiation. Conse-quently, chemical trapping experiments with 1-methyl-1-cyclohexene were set up for these dyes. The estimated 1O2yield for Thiac1 and Rhodac is about 0.005"20%, whereasfor Thiac2 and Thiac3 no 1O2 production could be demon-strated. Generally, it can be concluded that the singlet oxygenquantum yields of the thiacyanines are very low, in contrastto the indocyanines, which showed a somewhat higher singletoxygen quantum yield. The data for Thiac1 and Thiac4 arein agreement with a previous report [18].

4. Discussion

In view of the potential of cyanine dyes as new PDT tools,the present study was undertaken to evaluate the photode-pendent cytotoxic and antiproliferative characteristics of 35cyanine dyes. Initially, it was demonstrated that several thia-cyanines (five out of eight tested), indocyanines (four outof eight) and one rhodacyanine were photobiologicallyactive, i.e., they induced at least an additional 20% cytotoxic(using concentrations of 1 or 10 mM) or antiproliferative (1mM) effect after light irradition, compared with resultsobtained in dark conditions. Conversely, using the same cri-teria all oxa-, imida- and kryptocyanines tested proved not tobe photoactive. The latter observation is somewhat in contrastwith reports on the photocytotoxic characteristics of a numberof oxacyanines, indocyanines and kryptocyanines, includingthree non-photosensitizing dyes of the present study[3,24,25]. For instance, Oseroff et al. found that Indoc3 andOxac3 exhibited photoactivity on EJ bladder carcinoma cellsand also that indocyanine green (Indoc1) displayed a pho-totherapeutic effect on HaCaT cells [7]. Although differ-ences in photosensitivity between cell types cannot beexcluded, it should be stressed that very high fluences (e.g.,900 J/cm2 [3]) or high dye concentrations (e.g. up to 50mM [7]) were used in these studies. It is likely that the lessdrastic conditions employed in the present work did not allowphotobiological activity to be detected in some of the testeddyes. Conversely, it is anticipated that the dyes selected bythe present screening conditions are probably potent photo-sensitizers relevant to in vivo PDT.

Our results show that almost all the selected compoundsdisplayed a significant photosensitized increase in their cyto-toxic or antiproliferative effect. In particular, Rhodac dem-onstrated a potent photodependent cellular effect asexpressed

in the high ratios of CC50 values (Fc ratio) and IC50 values(FI ratio) (Table 3). It is of interest that another rhodacy-anine (i.e., MKT-077) has also exhibited a potent photode-pendent activity on mitochondrial respiration and on thestructural integrity of mitochondrial DNA [26]. Whether thishigh photosensitizing capacity is a general feature of rhoda-cyanines is presently unknown, since photophysical and pho-tobiological data on these rhodanine-based derivatives areseemingly lacking. However, straightforward syntheses forrhodacyanine analogues have been reported recently [27],and it will be a matter of time before more detailed infor-mation is available about this intriguing class of photosensi-tizing compounds. Several of them have already shown aspecific antitumoral effect in dark conditions [27].

In the present study, some parameters known to determinethe photoinduced cytotoxic and antiproliferative potency ofphotosensitizers were investigated. However, the degree ofcellular photosensitization neither correlated with the meas-ured molar absorption coefficients () nor with the singletoxygen quantum yields (Fs) of the respective compounds.For instance, Rhodac with a very high photosensitizationeffect exhibited a moderate value, while the opposite wastrue for Indoc2. The same discrepancies exist between thephoto-induced cellular effects and the singlet oxygen quan-tum yields: while Thiac1, Thiac2, Thiac3 and RhodacshowedFc ratios between 2.2 and 22 and FI ratios between 3.0 and11, all these dyes possess very low values of Fs. The indo-cyanines and Thiac5 showed about three- to tenfold higherFs values, but with Fc and FI ratios ranging from 1.1 to 3.5.As shown, the rather poor cellular photosensitization was notdue to a deficient light activation, since these compoundswere activated by the highest fluences used in this study.Only Thiac4 showed an increased singlet oxygen quantumyield in combination with high Fc and FI ratios (5.5 and 2.9,respectively). Furthermore it should be stressed that themeasured Fs values (ranging from 0.077 to-0.002) weresubstantially lower than those of the two standard photosen-sitizers used in this study (Table 2).

The latter data are remarkable, since in general it isassumed that a high 1O2 yield is one of the main features ofan efficient photosensitizer. However, low singlet oxygenquantum yields are typical for cyanines [6,7,18,2831] andcan be interpreted in terms of an efficient deactivation of theexcited cyanine dye via fluorescence, internal conversion andphotoisomerization [32]. Despite these poor photophysicalproperties, several cyanines (e.g., CY18, a trimethine car-bocyanine with long N-alkyl chains, MC540) produce potentphotosensitized cellular damage [32]. Furthermore, bymeasuring the relative phototoxicity of a collection of cati-onic dyes including cyanines, it was found that Thiac1 (butnot Thiac4) was 1000 times more phototoxic than could beexpected on basis of a 1O2 hypothesis [18].

To account for the potent photosensitizing effects of somecarbocyanines, in spite of their poor photophysical charac-teristics, it has been suggested that the diene structures of thedyes are likely to form oxydye intermediates after reaction



with photo-induced autogenerated singlet oxygen [18]. Thedegree of formation of these reactive intermediates wouldthen explain the extent of photo-induced cellular effects.Moreover, the Fs values of cyanines as assessed in a homo-geneous solution might underestimate the actual amount ofsinglet oxygen photogenerated in specific subcellular sites ofliving cells. For instance, it was found that MC540 andseveraldialkylthiacarbocyanines (but not Thiac1) produced signif-icantly higher amounts of 1O2 in model membrane systemsthan in an organic solvent due to a decreased mobility of thecompounds, hindering photoisomerization and internal con-version [30,33]. Interestingly, a specific localization of cya-nine dyes (e.g., at the inner mitochondrial membrane),allowing the compound to interact with a specific protein orstructure critical for cell viability, would also explain theinteresting photobiological activity of these dyes in spite oftheir poor photophysical characteristics [6,18]. In thisrespect, it should be mentioned that mitochondria are crucialmediators of an intrinsic pathway leading to programmed celldeath (apoptosis). For instance, recent findingshave revealedthat the release of mitochondrial cytochrome c into the cyto-sol, induced by many apoptogenic stimuli (including reactiveoxygen species (ROS)), constitutes the first step of a cascadeof events that ultimately lead to the activation of the apoptoticexecutioner caspase-3 [34]. The interaction of some cya-nines with mitochondrial constituents is presently underinvestigation on our laboratory.

Another important parameter in the determination of thecellular effects of photosensitizers is their extent of cellularaccumulation and subcellular distribution. All selected cati-onic compounds showed evident to prominent dark cellulareffects, probably due to an extensive accumulation in mito-chondria, causing impaired mitochondrial function and celldeath. Significantly, the only compound (Thiac5) that accu-mulated less than the other cationic dyes also showed areduced cytotoxic and antiproliferative effect. On the otherhand, Indoc2 exhibited an accumulation similar to Indoc4and Indoc7, but with a dramatically reduced antiproliferativeeffect. The net anionic character of this dye probably doesnot allow a mitochondrial accumulation to take place. Ourresults further corroborate the finding that thiacyanines withsmall N-alkyl groups (ethyl to decyl) are cytotoxic in thedark (e.g., Thiac1), while the longer-chain cyanines withhigh lipophilicity exhibit virtually no dark cellular effects[32]. In order to pass through the cellular membrane, a cat-ionic dye should possess a delocalized charge in combinationwith some lipophilicity [3]. Typically, a highly mitochon-dria-specific compound such as rhodamine 123 has anoctanol/water partition coefficient of 1.5 [3]. The presentstudy shows that cyanines with a partition coefficient closeto 1.5 accumulate to the highest extent, while the more hydro-phobic compounds (e.g., indocyanines) concentrate lessintracellularly.

Although the pronounced dark effects complicate a simpleinterpretation of the cellular photosensitization, a certaindegree of correlation seem to exist between the extent of

cellular accumulation and the cellular photodamage. Forinstance, compounds that were taken up very efficiently(Rhodac, Thiac3, Thiac4) were some of the best photosen-sitizers found in this study, while dyes that accumulated slug-gishly (Thiac5, Indoc2, Indoc7) behaved as poorphotosensitizers. However, the other compounds did not fol-low the same pattern and obviously no general rule can bededuced from the present results.

Collectively, our data show that eight cyanines, includingsix never described before, feature interesting photodepen-dent cytotoxic and antiproliferative effects. In particular,Rhodac showed very promising photodependent biologicalactivity. Future work will include a further optimization ofthe photophysical properties of some cyanines used in thisstudy, e.g., by introduction of structural modifications. Thismodification could include the incorporation of an internalheavy atom to facilitate intersystem crossing and henceincrease the 1O2 quantum yield, for instance by the substitu-tion of tellurium for sulfur in cyanine rings [28]. Further-more, some of the investigated compounds will be used forfurther exploration of their in vivo antitumor activity.

5. Abbreviations

BSA bovine serum albuminDMSO dimethylsulfoxideEDKC N,N9-bis(2-ethyl-1,3-dioxolane)-

kryptocyanineFBS fetal bovine serumHPD hematoporphyrin derivativeMC540 merocyanine 540MEM Minimum Essential MediumPBS phosphate-buffered salinePDT photodynamic therapy

Acknowledgements

The authors thank Dr F. De Schryver for his critical reviewof the manuscript and helpful comments. This work wassupported by grants awarded by Fonds voor Wetenschap-pelijk Onderzoek-Vlaanderen (F.W.O.-Vlaanderen) and bya grant (Onderzoekstoelage) awarded by the K.U.Leuven.F.v.L. is a recipient of a fellowship from the Vlaams Instituutvoor Bevordering van Wetenschappelijk-TechnologischOnderzoek in de Industrie (I.W.T.). D.DeV. is a researchleader with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.

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A Comparative Study of the Photosensitizing Characteristics of Some Cyanine Dyes

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