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J. Photochem. Photobiol. B: Biol. 55 (2000) 2736
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
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
Article: 7933
28
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.
Article: 7933
studies were performed to correlate the photocytotoxic activity of the cyanines with their singlet oxygen quantum yield.
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
Article: 7933
30
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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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-
Article: 7933
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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
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
Article: 7933
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).
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).
Article: 7933
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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
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
Article: 7933
35
5. Abbreviations
BSA
DMSO
EDKC
FBS
HPD
MC540
MEM
PBS
PDT
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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