A comparative study of the photosensitizing characteristics of some cyanine dyes

www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 55 (2000) 27–36

A comparative study of the photosensitizing characteristics of some 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 the krypto-, oxa- and imidacyanines is photobiologically active on HeLa cells. Conversely, five thiacyanines (Thiac1–5), one rhodacyanine (Rhodac) and four indocyanines (Indoc2, Indoc4, Indoc5, Indoc7) show photodependent cytotoxicity or antiproliferative effects. A more detailed study shows that out of the ten selected compounds, eight cyanines feature significant photodependent cytotoxic and antiproliferative effects. All possess maximum absorption ranges between 545 and 824 nm. In particular, Rhodac, a tetramethinemeromonomethine rhodacyanine dye with an absorption maximum of 655 nm (ethanol) and a molar absorption coefficient ´s108 000 shows very promising photodependent biological activity. In general, the measured singlet oxygen quantum yield of the selected cyanines is low (-0.08) and does not correlate with the degree of photosensitization. Furthermore, the present study shows that cyanines with a partition coefficient close to 1.5 accumulate to the highest extent in HeLa cells, while the more hydrophobic compounds (e.g., indocyanines) concentrate less intracellularly. q2000 Elsevier Science S.A. All rights reserved. Keywords: Carbocyanines; Photosensitization; Cytotoxicity; HeLa; Cellular uptake

1. Introduction Photodynamic therapy (PDT) is an alternative modality in cancer treatment and is based on the use of photosensitizing chemicals that preferentially accumulate in target tumor cells. Photofrin, which is a synthetic hematoporphyrin derivative (HPD), is commonly used in clinical trials for PDT of various cancers. Although this HPD is efficacious and safe in the treatment of different human cancers, it has several disadvantages, such as its complex chemical composition, the long retention time in several types of normal tissue (about 4–6 weeks) and weak absorbance above 600 nm [1]. Therefore, new photosensitizers are being developed with increased chemical purity, low dark systemic toxicity, strong absorption in the phototherapeutic window from 600 to 1000 nm and preferential tumor localization [2]. So far, no single photosensitizer is available that exhibits all of these properties, and the 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 during the last decade [3,4]. The economic interest in these dyes goes back to their effectiveness as photographic sensitizers and numerous compounds are commercially available. Cyanines consist of two heterocycles linked by a monomethine bridge, while carbocyanines and dicarbocyanines contain an oligomethine bridge (Figs. 1 and 2). Resonance between the ring systems creates chromophores that absorb in the visible region, frequently with high molar absorption coefficients. Most of these compounds are cationic, in contrast to the more frequently used anionic photosensitizers such as hematoporphyrin derivatives, chlorins and sulfonated phthalocyanines. Since typically an electrical potential gradient of about y180 mV exists across the mitochondrial membrane, cationic cyanines strongly concentrate into mitochondria, up to 1000-fold with respect to the extracellular concentration [3]. More specifically, it has been found that cationic dyes such as EDKC (N,N9-bis(2-ethyl-1,3-dioxolane)kryptocyanine) are retained in vitro and in vivo to a much greater extent in the mitochondria of carcinoma and melanoma cells than in nor-

1011-1344/00/$ - see front matter q2000 Elsevier Science S.A. All rights reserved. PII S 1 0 1 1 - 1 3 4 4 ( 0 0 ) 0 0 0 2 1 - X

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

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

However, cyanines featuring a net anionic charge due to the presence of sulfonate substituents have also been used as selective and effective photosensitizers. For instance, merocyanine 540 (MC540), a negatively charged oxacyanine derivative, is used for the selective purging of ocular leukemia, lymphoma and neuroblastoma cells in autologous bone marrow grafts. It is likely that the compound predominantly functions as a membrane-bound 1O2 photogenerator [4,6]. More recently indocyanine green [7,8], an anionic indotricarbocyanine derivative, was also introduced into photomedicinal practice [7,8]. In this study, we have analyzed the photodynamic action of 35 cyanines, belonging to several chemical subgroups, on HeLa cells. Furthermore, the in vitro photocytotoxic and antiproliferative effect of the photoactive cyanines was studied in more detail. In addition, the cellular accumulation in HeLa cells and the partition coefficient of the selected cyanines were investigated in order to elucidate the background of the observed dark and photodependent effects. Finally, ESR

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

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studies were performed to correlate the photocytotoxic activity 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 (Milwaukee, WI, USA) and used as received. Hypericin was prepared as described earlier [9,10]. The compounds were dissolved in dimethylsulfoxide (DMSO) for the cell-culture experiments and stored at y208C in dark conditions. Under these conditions, the solutions were stable for more than two months. Photofrin (Ispen Pharma, Ettlingen, Germany) was reconstituted in 5% dextrose in water at a concentration of 25 mg/ml just before use in accordance with the manufacturer’s instructions. HeLa cells (cervix carcinoma, human) were obtained from American Type Culture Collection (Rockville, MD, USA). The cells (passage range between 10 and 30) were grown at 378C in a humidified 5% CO2 and 95% air atmosphere in Minimum Essential Medium (MEM) with Earle’s salts containing 2 mM L-glutamine, non-essential amino acids (100=), penicillin (100 IU/ml), streptomycin (100 mg/ml), tylocine (60 mg/ml), amphotericin B (0.25 mg/ml) and 10% fetal bovine serum (FBS). All culture-medium compounds were purchased from Gibco BRL (Paisley, Scotland). Dilutions of stock solutions were made in cell-culture medium, with a final DMSO concentration of 0.1%. 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 halogen lamp (Philips) with a prominent spectral output in the absorption region of the different dyes (Fig. 3(a)). At the surface of the plates, the uniform fluence rate was 23 mW/ cm2, as measured with an IL 1400 radiometer (International Light, Newburyport, MA). The cells were irradiated for 15 min. During irradiation, the temperature never exceeded 298C. This temperature did not influence the viability of the cells. Visible spectra were recorded in ethanol on an Ultrospec 2000 UV–Vis spectrophotometer (Pharmacia, Uppsala, Sweden). 2.3. Cytotoxicity assay The photocytotoxic effect 1 h after irradiation was determined by assessing cell viability using Neutral Red (Acros, Geel, Belgium), a known specific marker for lysosomal integrity [11]. HeLa cells were seeded onto 96-well tissueculture plates at 3=104 cells per well, and were incubated for 24 h at 378C. The medium was replaced under strictly subdued light conditions (-1 mW/cm2), with fresh medium containing different concentrations of dye or DMSO. Sub-

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Table 1 Product name or product number of oxacyanines, thiacyanines, rhodacyanine, indocyanines, imidacyanines and kryptocyanine and derivatives (available from Aldrich and Sigma–Aldrich Library of rare chemicals structure index) used in the present study to screen for photocytotoxic compounds. After selection, the marked substances (U) were studied in more detail. The products are preceded by the code names that are used in this paper for convenience. Oxacyanines Oxac1 Oxac2 Oxac3 Oxac4

Thiacyanines Thiac1 Thiac2 Thiac3

Thiac4 Thiac5 Thiac6 Thiac7 Thiac8 Rhodacyanine Rhodac

Indocyanines Indoc1 Indoc2 Indoc3 Indoc4 Indoc5 Indoc6 Indoc7 Indoc8 Imidacyanines Imidac1 Imidac2 Imidac3 Imidac4 Imidac5 Imidac6

Kryptocyanines Kryptoc1 Kryptoc2 Kryptoc3 Kryptoc4 Kryptoc5 Kryptoc6 Kryptoc7 Kryptoc8

3,39-diethyloxacarbocyanine iodide 3,39-diethyloxadicarbocyanine iodide 3,39-diethyloxatricarbocyanine iodide ethyl-(ethyl-(ethyl-phenyl-benzooxazol-yl)buta-dienyl)-phenyl-benzooxazol-3-ium ethane sulfonate 3,39-diethylthiacarbocyanine iodide (U) 3,39-diethyl-9-methylthiacarbocyanine iodide (U) 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2ylidene)-2-methylpropenyl]-naphtho[1,2-d]thiazolium bromide (U) 3,39-diethylthiadicarbocyanine iodide (U) 3,39-diethylthiatricarbocyanine iodide (U) (hydroxy-ethyl)-(((hydroxy-ethyl)-benzothiazolylidene)-methyl-propenyl)-benzothiazolium chloride RCL S17,346-0 RCL S17,348-7 5-[3-ethoxy-4-(3-ethyl-5-methyl-2(3H)benzothiazolylidene)-2-butenylidene]-3-ethyl-2-[(3-ethyl-4,5-diphenyl-2(3H)-thiazolylidene)methyl]-4,5-dihydro-4oxothiazolium iodide (U) indocyanine green new indocyanine green (U) 1,19,3,3,39,39-hexamethyl-indodicarbocyanine iodide 1,19,3,3,39,39-hexamethyl-indotricarbocyanine iodide (U) IR-768 perchlorate (U) IR-780 iodide IR-792 perchlorate (U) IR-1048 RCL S13,111-3 RCL S17,120-4 RCL S17,123-9 RCL S17,132-8 direct red 39 5-cyano-2-[3-(5-cyano-1,3-diethyl-1,3-dihydro-2Hbenzimidazol-2-ylidene)-1-propenyl]-1-ethyl-3-(4sulfobutyl)-1H-benzimidazolium hydroxide, inner salt 1,19-diethyl-2,29-cyanine iodide 1,19-diethyl-2,49-cyanine iodide 1,19-diethyl-4,49-cyanine iodide 1,19-diethyl-2,29-carbocyanine iodide 1,19-diethyl-4,49-carbocyanine iodide 1,19-diethyl-4,49-dicarbocyanine iodide 1,19-diethyl-2,29-dicarbocyanine iodide 1,19-diethyl-2,29-quinotricarbocyanine iodide

sequently, the cells were incubated under dark conditions at 378C for 24 h. Under these conditions, all the cyanines were stable. The drug-containing medium was then replaced with

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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 different compounds (b). Spectra in (b) were normalized at their highest peak.

drug-free medium under subdued light conditions after washing with phosphate-buffered saline (PBS), and cells were immediately light irradiated (or not, in the case of ‘dark’ cytotoxicity). Afterwards cells were incubated at 378C under dark conditions for 45 min. The amount of Neutral Red accumulated in the viable cells was measured at 550 nm using a microtiter plate reader (SLT, Salzburg, Austria) and expressed as the percentage of dye extracted from untreated control cells. After curve fitting using non-linear regression (Prism, San Diego, CA, USA), CC50 values (the concentration giving 50% cytotoxicity in comparison with the control) were determined for each experiment. The mean CC50 value was calculated from three independent experiments. 2.4. Antiproliferative assay The antiproliferative assay using HeLa cells was performed as reported in Ref. [10]. Briefly, 1=103 cells per well were seeded onto 96-well plates, and incubated for 24 h at 378C. Subsequently, the cells were washed with PBS and incubated with dye or DMSO for 24 h at 378C, and irradiated

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or not. After the irradiation, cells were incubated under dark conditions for three days. Cell proliferation was determined by quantification of the cellular protein content using Naphthol Blue Black (Acros, Geel, Belgium), according to the method of Palombella and Vilcek [12]. The amount of dye was measured at 620 nm using a microtiter plate reader. After curve fitting using non-linear regression (Prism), the IC50 values (the concentration resulting in 50% inhibition of cell proliferation when compared with the control) were determined for each experiment (ns3). 2.5. Cellular accumulation HeLa cells (9=105) were plated and incubated for 48 h in six-well tissue culture plates (Falcon). They were then exposed to 1 or 5 mM dye (or DMSO) for 24 h under dark conditions. Subsequently, the cells were rinsed under subdued light conditions twice with PBS containing 2% bovine serum albumin (BSA, Fluka, Buchs, Switzerland) and twice with PBS. Subsequently, 1 ml trypsin (Gibco BRL, Paisley, Scotland) was added to the cells and they were scraped from

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the bottom of the culture plate with a rubber policeman. The number of cells recovered per well was determined using a cell counter (Coulter Z1 Particle Counter, Coulter Electronic, UK) in all experiments. The cell suspension was then centrifuged (6000g, 10 min) and the dyes were extracted from the cell pellet with 500 ml DMSO under sonication for 2 min. After centrifugation (6000g, 10 min), the content of Thiac1– 4 and Rhodac present in DMSO was analysed with a microplate fluorescence reader (FL600, Bio-tek, Winooski, VT, USA) using calibration curves. The excitation and emission wavelengths were set at 590 and 645 nm for Thiac3, Thiac4 and Rhodac and at 530 and 590 nm for Thiac1–2. The quantification of the other dyes in DMSO was performed by measuring the absorbance of the supernatants containing the dyes at lmax. Dye concentrations were calculated by a calibration curve. The experiment was done in triplicate.

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al. [16]. 1O2 quantum yields were calculated using Rose Bengal as a reference (Fss0.76). The method was controlled by measuring the 4-oxo-TEMPO production rate and calculating the 1O2 quantum yields for hypericin (0.73"0.03) and Methylene Blue (0.25"0.03), which were in correspondence with literature values. When the 1O2 formation was too weak to be detected with the 4-oxo-TEMP reaction, chemical trapping experiments were set up with 1-methyl-1-cyclohexene (Acros) (2 mmol 1-methyl-1-cyclohexene in 2 ml ethanol, 15 mM sensitizer, 24 h irradiation) [17]. 2.8. Statistical analysis The significance of differences was calculated by using Student’s t-test. Values of p-0.05 were considered to be significant.

2.6. Determination of partition coefficients n-Octanol was purified by shaking with 1N sodium hydroxide, followed by washing with water until neutral. Partition coefficients were determined by adding 20 mM of each compound to 5 ml octanol and an equal volume of PBS (pH 7.4) was added. Each phase was presaturated with the other. The tubes were then vortexed for 1 min at high speed and placed in a shaker for 4 h at room temperature (21– 228C), followed by centrifugation for 10 min at approximately 500g to separate the octanol and water phase. The phase containing the larger quantity of the compound being partitioned (octanol) was then removed and added to a fresh aliquot of the opposite phase, and reprocessed as described above. This procedure was repeated twice in order to stabilize the partition coefficient as described in Ref. [13]. The drug concentrations in the octanol and in the aqueous phase were measured from UV absorbance and deduced from the standard curve. The whole procedure was performed in subdued light conditions. 2.7. ESR Photosensitized singlet molecular oxygen (1O2) yields were determined by trapping of 1O2 with 2,2,6,6-tetramethyl4-piperidone (4-oxo-TEMP), and measuring the concentration of the formed 2,2,6,6-tetramethyl-4-piperidone-N-oxyl radical (4-oxo-TEMPO) with ESR [14,15]. X-band ESR spectra (microwave power, 20 mW; modulation amplitude, 3.16 G; 100 kHz field modulation) were recorded with a Bruker ESP-300 apparatus at room temperature. Samples (150 ml) were injected into a flat ESR quartz cell and illuminated directly inside the microwave cavity using a Schott KL-1500 cold light source. 4-oxo-TEMP was purchased from Acros (Geel, Belgium) and used as received. Typically the reaction mixtures consist of an oxygen-saturated ethanol solution containing the sensitizer (30 mM) and 4-oxo-TEMP (0.1 M). Spin trapping of 1O2 by 4-oxo-TEMP was performed according to a modification of the method of Lion et

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3. Results 3.1. Photodependent cytotoxic and antiproliferative effects In a preliminary set of experiments, the photodependent and dark cytotoxic and antiproliferative effects of four oxacyanines, eight thiacyanines, one rhodacyanine, eight indocyanines, six imidacyanines, one kryptocyanine and seven derivatives (Table 1) were tested on HeLa cells. Hypericin and Photofrin were included as standard photosensitizers. All cyanine compounds feature a positive charge, except for Imidac1, Imidac3 and Imidac6, which are neutral, and Indoc1, Indoc2 and Imidac2, which are anionic compounds due to the presence of one or two sulfonate groups, respectively (Table 1). Since all dyes showed a large variety of absorption spectra in the visible spectrum (see below), a lamp with a broad and continuous spectrum was chosen. The spectral output of the lamp is shown in Fig. 3(a). HeLa cells were incubated with 1 and 10 mM of each of the compounds and irradiated (or not) with light after 24 h. The photocytotoxic effect 1 h after irradiation was determined by assessing cell viability using Neutral Red. Photocytotoxicity tested in this way therefore assesses early photodependent damage to subcellular organelles. Alternatively, the long-term cellular effect of the photoactivated compounds (1 mM) was tested in an antiproliferation assay. Compounds were considered to exhibit photobiological activity only when the cytotoxicity (at the 1 or 10 mM level) or antiproliferative effect (at the 1 mM level) increased by at least an additional 20% after light irradition, as compared with the results obtained in dark conditions. Using these criteria, it was found that none of the krypto-, oxa- or imidacyanines was photobiologically active on HeLa cells. Conversely, five cationic thiacyanines (Thiac1-5), one cationic rhodacyanine (Rhodac), one anionic (Indoc2) and three cationic (Indoc4, Indoc5, Indoc7) indocyanines (Figs. 1 and 2) showed photodependent cytotoxicity or antiproli-

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

lmax (nm)

´ (My1 cmy1)

Fs a

LogPo/w

Thiac1 Thiac2 Thiac3 Thiac4 Thiac5 Rhodac Indoc2 Indoc4 Indoc5 Indoc7 Hypericin Photofrin

560 545 575 655 764 655 824 742 771 797 598 630

144000 86000 80000 232000 199000 108000 1466000 231000 276000 264000 39500 3200

0.005"0.001 -0.002 -0.002 0.033 0.013 0.005"0.001 0.077 0.054 0.027 0.011 0.73"0.03 0.89 c

1.85 2.11 1.82 1.94 2.10 1.74 n.d. b 2.39 2.22 2.21 n.d. b n.d. b

a

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

ferative effects. The latter compounds, as well as the standard photosensitizers hypericin and Photofrin, were studied in more detail. The absorption spectra of the cyanines and the maximum absorption wavelength as well as the molar absorption coefficients of the photosensitizers used are collected in Table 2 and Fig. 3(b). Since the spectral output of the lamp is somewhat variable, the extent of overlap of the absorption spectrum and the lamp emission was taken into account to understand correctly the photosensitizing effect of each of the dyes used. For that purpose, the spectral output of the lamp was integrated over the area defined by the different absorption peaks (e.g., 600–900 nm for Indoc2, see Fig. 3(b)) and the corresponding fluences were calculated (Table

3). In the cases of hypericin and Photofrin, which exhibit several peaks in the visible spectrum (data not shown), the range 400–650 nm was used to calculate the respective fluences. From Table 3 it can be seen that the difference between the minimal fluence (3 J/cm2) and the maximal fluence (12 J/cm2) used is a factor of four. For each of the selected dyes, the CC50 value (the concentration giving 50% cytotoxicity in comparison with the control) and IC50 value (the concentration resulting in 50% inhibition of cell proliferation) determined in dark conditions and after light activation were determined. The data show that all of the selected cyanines, except for Indoc2 and Indoc7, exhibited a significant photodependent cytotoxic or antiproliferative effect, as indicated by the ratio of both the CC50 values (Fc) and the IC50 values (FI) (Table 3). No correlation between the calculated fluence and the ratio values was found, demonstrating that the limited photoeffect displayed by a few compounds was not due to their deficient photoactivation. The results for Thiac1 and Thiac4 are consistent with previously reported data [18]. As can be noticed further, IC50 values are about one order of magnitude lower than the corresponding CC50 values for the cyanines, except for Indoc2, which showed similar IC50 and CC50 values. However, the Fc and FI values obtained in dark and light conditions are approximately the same, except for Thiac1. This suggests that for most of the compounds, the degree of photodamage on top of the effects induced in dark conditions is similar for cytotoxicity and antiproliferative effects. Although structural differences are limited among Thiac1– 4 and Thiac5 (Fig. 2), the least active, marked variations exist between their photodependent and dark cytotoxic and antiproliferative effects. In the case of Rhodac, a complex tetramethinemeromonomethine rhodacyanine dye containing

Table 3 The cytotoxic and antiproliferative effects of the photosensitizers used on HeLa cells determined in dark conditions or after light activation CC50 (mM) a

Thiac1 Thiac2 Thiac3 Thiac4 Thiac5 Rhodac Indoc2 Indoc4 Indoc5 Indoc7 Hypericin Photofrin

Fc b

light

dark

0.41"0.2 2.1"1 0.5"0.04 0.64"0.3 4.5"0.8 0.39"0.1 2.1"0.5 )10 0.60"0.3 0.94"0.3 0.83"0.1 5.82"0.27 e

9.1"0.2UUU 6.2"2U 1.1"0.3U 3.5"0.5UU 6.5"2.2 4.2"1.3UU 3.1"1 )10 2.1"0.5U 1.0"0.2 )10 )25 e

22 3.0 2.2 5.5 1.4 11 1.5 – 3.5 1.1 )12 )4.3

IC50 (mM) a

FI c

light

dark

0.051"0.01 0.066"0.03 0.032"0.009 0.059"0.02 0.45"0.05 0.055"0.02 2.0"0.9 0.29"0.1 0.061"0.01 0.29"0.04 0.23"0.08 2.57"0.12 e

0.19"0.04UU 0.20"0.1 0.097"0.01UU 0.17"0.08 0.80"0.2U 0.63"0.16UU 2.2"1.0 0.69"0.1UU 0.20"0.07U 0.41"0.09 )10 )25 e

Fluence d (J/cm2)

3.7 3.0 3.0 2.9 1.8 11 1.1 2.4 3.3 1.4 )43 )10

3 3 4 5 10 6 12 9 9 11 5 5

Means"SD of independent experiments (ns3) are given. The mean"SD of the dark conditions was statistically analyzed versus light conditions (Up-0.05, p-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. a

UU

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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 (filled bars) for 24 h. Bars represent the mean"SD of different experiments (ns3).

a 4-oxothiazolidine ring (rhodanine) bridging two thiazole heterocycles, the ratio between the dark and light-dependent cytotoxic and antiproliferative effects was as high as 11. In this case the calculated fluence was 6 J/cm2, which is close to the fluence used to activate the standard photosensitizers hypericin and Photofrin (5 J/cm2). In general, the data for the indocyanines are consistent with those for the thiacyanines, except for the anionic Indoc2, which displayed an antiproliferative effect only in the micromolar range. Hypericin and Photofrin, both potent standard photosensitizers, exhibited CC50 values of 0.83 mM and 5.8 mg/ml and IC50 values of 0.23 mM and 2.57 mg/ml, respectively (Table 3). As can be seen further, these compounds showed substantially lower cytotoxic and antiproliferative effects in dark conditions than the cyanines. These data are in line with previous data on their cytotoxic effects after photoactivation [10,19–22] and further prove the validity of the experimental set-up used in the present work to screen for photoactive cyanines.

cellular concentrations of 1 and 5 mM, respectively. Surprisingly, the intracellular concentration of Thiac5, a homologue of Thiac1 and Thiac4, could not be determined due to values lower than the detection limit (-10 mM). In general, indocyanine dyes accumulated in the cells to a somewhat lower extent. As revealed by the octanol/water partition coefficients (Table 2, PO/W), all cationic compounds displayed hydrophobic behavior. It can be seen that cyanines with a relatively low PO/W were concentrated to a larger extent in the cells than those with a higher partition coefficient. In general, a linear correlation (R2: 0.68; p-0.01) was found between the logarithm of the intracellular concentration and the logarithm of PO/W (Fig. 5) when the cells were incubated with 5 mM dye. However, in case of a 1 mM extracellular concentration, the correlation coefficient was somewhat lower (R2: 0.54; p-0.04) (results not shown).

3.2. Cellular accumulation Cellular accumulation and partition coefficients of the selected cyanines were then investigated to explore further the background of the observed dark and photodependent effects. For the cellular accumulation experiments, HeLa cells were incubated with the selected cyanines (1 and 5 mM) for 24 h. Afterwards the trypsinized cells were counted, and the dye was extracted and quantified. The final intracellular concentration was converted to units of mM assuming 3 ml as the mean volume of 106 cells [23]. It can be seen that Thiac3, Thiac4 and Rhodac accumulated dramatically more in HeLa cells than the other compounds (Fig. 4). In the case of Rhodac, average intracellular concentrations as high as 531 ("55) and 1674 ("18) mM were obtained using extra-

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

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3.3. Singlet oxygen quantum yield In order to determine the singlet oxygen quantum yields of the selected cyanines and hypericin, 4-oxo-TEMP was reacted with 1O2 to generate the stable radical 4-oxo-TEMPO, which can easily be detected with ESR [14,15]. 4-oxoTEMPO formation is directly related to the 1O2-generating quantum yields. The singlet oxygen quantum yields of the photosensitizers used are mentioned in Table 2. For Thiac1, Thiac2, Thiac3 and Rhodac, the formation of 4-oxo-TEMPO could not be detected with ESR upon irradiation. Consequently, chemical trapping experiments with 1-methyl-1cyclohexene were set up for these dyes. The estimated 1O2 yield for Thiac1 and Rhodac is about 0.005"20%, whereas for Thiac2 and Thiac3 no 1O2 production could be demonstrated. Generally, it can be concluded that the singlet oxygen quantum yields of the thiacyanines are very low, in contrast to the indocyanines, which showed a somewhat higher singlet oxygen quantum yield. The data for Thiac1 and Thiac4 are in 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 photodependent cytotoxic and antiproliferative characteristics of 35 cyanine dyes. Initially, it was demonstrated that several thiacyanines (five out of eight tested), indocyanines (four out of eight) and one rhodacyanine were photobiologically active, i.e., they induced at least an additional 20% cytotoxic (using concentrations of 1 or 10 mM) or antiproliferative (1 mM) effect after light irradition, compared with results obtained in dark conditions. Conversely, using the same criteria all oxa-, imida- and kryptocyanines tested proved not to be photoactive. The latter observation is somewhat in contrast with reports on the photocytotoxic characteristics of a number of oxacyanines, indocyanines and kryptocyanines, including three non-photosensitizing dyes of the present study [3,24,25]. For instance, Oseroff et al. found that Indoc3 and Oxac3 exhibited photoactivity on EJ bladder carcinoma cells and also that indocyanine green (Indoc1) displayed a phototherapeutic effect on HaCaT cells [7]. Although differences in photosensitivity between cell types cannot be excluded, it should be stressed that very high fluences (e.g., 900 J/cm2 [3]) or high dye concentrations (e.g. up to 50 mM [7]) were used in these studies. It is likely that the less drastic conditions employed in the present work did not allow photobiological activity to be detected in some of the tested dyes. Conversely, it is anticipated that the dyes selected by the present screening conditions are probably potent photosensitizers relevant to in vivo PDT. Our results show that almost all the selected compounds displayed a significant photosensitized increase in their cytotoxic or antiproliferative effect. In particular, Rhodac demonstrated a potent photodependent cellular effect as expressed

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in the high ratios of CC50 values (Fc ratio) and IC50 values (FI ratio) (Table 3). It is of interest that another rhodacyanine (i.e., MKT-077) has also exhibited a potent photodependent activity on mitochondrial respiration and on the structural integrity of mitochondrial DNA [26]. Whether this high photosensitizing capacity is a general feature of rhodacyanines is presently unknown, since photophysical and photobiological data on these rhodanine-based derivatives are seemingly lacking. However, straightforward syntheses for rhodacyanine analogues have been reported recently [27], and it will be a matter of time before more detailed information is available about this intriguing class of photosensitizing compounds. Several of them have already shown a specific antitumoral effect in dark conditions [27]. In the present study, some parameters known to determine the photoinduced cytotoxic and antiproliferative potency of photosensitizers were investigated. However, the degree of cellular photosensitization neither correlated with the measured molar absorption coefficients (´) nor with the singlet oxygen quantum yields (Fs) of the respective compounds. For instance, Rhodac with a very high photosensitization effect exhibited a moderate ´ value, while the opposite was true for Indoc2. The same discrepancies exist between the photo-induced cellular effects and the singlet oxygen quantum yields: while Thiac1, Thiac2, Thiac3 and Rhodac showed Fc ratios between 2.2 and 22 and FI ratios between 3.0 and 11, all these dyes possess very low values of Fs. The indocyanines and Thiac5 showed about three- to tenfold higher Fs values, but with Fc and FI ratios ranging from 1.1 to 3.5. As shown, the rather poor cellular photosensitization was not due to a deficient light activation, since these compounds were activated by the highest fluences used in this study. Only Thiac4 showed an increased singlet oxygen quantum yield in combination with high Fc and FI ratios (5.5 and 2.9, respectively). Furthermore it should be stressed that the measured Fs values (ranging from 0.077 to-0.002) were substantially lower than those of the two standard photosensitizers used in this study (Table 2). The latter data are remarkable, since in general it is assumed that a high 1O2 yield is one of the main features of an efficient photosensitizer. However, low singlet oxygen quantum yields are typical for cyanines [6,7,18,28–31] and can be interpreted in terms of an efficient deactivation of the excited cyanine dye via fluorescence, internal conversion and photoisomerization [32]. Despite these poor photophysical properties, several cyanines (e.g., CY18, a trimethine carbocyanine with long N-alkyl chains, MC540) produce potent photosensitized cellular damage [32]. Furthermore, by measuring the relative phototoxicity of a collection of cationic dyes including cyanines, it was found that Thiac1 (but not Thiac4) was 1000 times more phototoxic than could be expected on basis of a 1O2 hypothesis [18]. To account for the potent photosensitizing effects of some carbocyanines, in spite of their poor photophysical characteristics, it has been suggested that the diene structures of the dyes are likely to form oxy–dye intermediates after reaction

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with photo-induced autogenerated singlet oxygen [18]. The degree of formation of these reactive intermediates would then explain the extent of photo-induced cellular effects. Moreover, the Fs values of cyanines as assessed in a homogeneous solution might underestimate the actual amount of singlet oxygen photogenerated in specific subcellular sites of living cells. For instance, it was found that MC540 and several dialkylthiacarbocyanines (but not Thiac1) produced significantly higher amounts of 1O2 in model membrane systems than in an organic solvent due to a decreased mobility of the compounds, hindering photoisomerization and internal conversion [30,33]. Interestingly, a specific localization of cyanine dyes (e.g., at the inner mitochondrial membrane), allowing the compound to interact with a specific protein or structure critical for cell viability, would also explain the interesting photobiological activity of these dyes in spite of their poor photophysical characteristics [6,18]. In this respect, it should be mentioned that mitochondria are crucial mediators of an intrinsic pathway leading to programmed cell death (apoptosis). For instance, recent findings have revealed that the release of mitochondrial cytochrome c into the cytosol, induced by many apoptogenic stimuli (including reactive oxygen species (ROS)), constitutes the first step of a cascade of events that ultimately lead to the activation of the apoptotic executioner caspase-3 [34]. The interaction of some cyanines with mitochondrial constituents is presently under investigation on our laboratory. Another important parameter in the determination of the cellular effects of photosensitizers is their extent of cellular accumulation and subcellular distribution. All selected cationic compounds showed evident to prominent dark cellular effects, probably due to an extensive accumulation in mitochondria, causing impaired mitochondrial function and cell death. Significantly, the only compound (Thiac5) that accumulated less than the other cationic dyes also showed a reduced cytotoxic and antiproliferative effect. On the other hand, Indoc2 exhibited an accumulation similar to Indoc4 and Indoc7, but with a dramatically reduced antiproliferative effect. The net anionic character of this dye probably does not allow a mitochondrial accumulation to take place. Our results further corroborate the finding that thiacyanines with small N-alkyl groups (ethyl to decyl) are cytotoxic in the dark (e.g., Thiac1), while the longer-chain cyanines with high lipophilicity exhibit virtually no dark cellular effects [32]. In order to pass through the cellular membrane, a cationic dye should possess a delocalized charge in combination with some lipophilicity [3]. Typically, a highly mitochondria-specific compound such as rhodamine 123 has an octanol/water partition coefficient of 1.5 [3]. The present study shows that cyanines with a partition coefficient close to 1.5 accumulate to the highest extent, while the more hydrophobic compounds (e.g., indocyanines) concentrate less intracellularly. Although the pronounced dark effects complicate a simple interpretation of the cellular photosensitization, a certain degree of correlation seem to exist between the extent of

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35

cellular accumulation and the cellular photodamage. For instance, compounds that were taken up very efficiently (Rhodac, Thiac3, Thiac4) were some of the best photosensitizers found in this study, while dyes that accumulated sluggishly (Thiac5, Indoc2, Indoc7) behaved as poor photosensitizers. However, the other compounds did not follow the same pattern and obviously no general rule can be deduced from the present results. Collectively, our data show that eight cyanines, including six never described before, feature interesting photodependent cytotoxic and antiproliferative effects. In particular, Rhodac showed very promising photodependent biological activity. Future work will include a further optimization of the photophysical properties of some cyanines used in this study, e.g., by introduction of structural modifications. This modification could include the incorporation of an internal heavy atom to facilitate intersystem crossing and hence increase the 1O2 quantum yield, for instance by the substitution of tellurium for sulfur in cyanine rings [28]. Furthermore, some of the investigated compounds will be used for further exploration of their in vivo antitumor activity.

5. Abbreviations BSA DMSO EDKC FBS HPD MC540 MEM PBS PDT

bovine serum albumin dimethylsulfoxide N,N9-bis(2-ethyl-1,3-dioxolane)kryptocyanine fetal bovine serum hematoporphyrin derivative merocyanine 540 Minimum Essential Medium phosphate-buffered saline photodynamic therapy

Acknowledgements The authors thank Dr F. De Schryver for his critical review of the manuscript and helpful comments. This work was supported by grants awarded by ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’ (F.W.O.-Vlaanderen) and by a grant (Onderzoekstoelage) awarded by the K.U.Leuven. F.v.L. is a recipient of a fellowship from the ‘Vlaams Instituut voor Bevordering van Wetenschappelijk-Technologisch Onderzoek in de Industrie’ (I.W.T.). D.DeV. is a research leader with the ‘Fonds voor Wetenschappelijk OnderzoekVlaanderen’.

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