www.elsevier.

nl/locate/jphotobiol
J. Photochem. Photobiol. B: Biol. 55 (2000) 2736

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 (Thiac15), 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 46
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: peter.dewitte@farm.kuleuven.ac.be

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

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 manufacturers 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 Earles 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 UVVis 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-

Wednesday May 03 09:52 AM

29

Table 1
Product name or product number of oxacyanines, thiacyanines, rhodacyanine, indocyanines, imidacyanines and kryptocyanine and derivatives
(available from Aldrich and SigmaAldrich 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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E. Delaey et al. / J. Photochem. Photobiol. B: Biol. 55 (2000) 2736

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

Wednesday May 03 09:52 AM

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 Thiac12. 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.

31

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
Students 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

Wednesday May 03 09:52 AM

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

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

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., 600900 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 400650 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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33

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 [10,1922] 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-

Wednesday May 03 09:52 AM

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

Wednesday May 03 09:52 AM

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 [6,7,18,2831] 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 oxydye intermediates after reaction

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

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

Wednesday May 03 09:53 AM

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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