In vitro toxicity testing of supramolecular sensitizers for photodynamic therapy

Toxicology in Vitro 17 (2003) 775–778 www.elsevier.com/locate/toxinvit

In vitro toxicity testing of supramolecular sensitizers for photodynamic therapy H. Kola´rova´a,*, J. Mosingerb, R. Lenobela, K. Kejlova´c, D. Jı´rova´d, M. Strnada a

Department of Biophysics, Faculty of Medicine, Laboratory of Growth Regulators, Palacky University, Hnı`votı´nska´ 3, 775 15 Olomouc, Czech Republic b Department of Inorganic Chemistry, Faculty of Sciences, Charles University in Prague, Prague, Czech Republic c National Institute of Public Health, Prague, Czech Republic d Faculty of Social and Health Studies, University of South Bohemia, Czech Republic Accepted 30 June 2003

Abstract We report the phototoxicity of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) and zinc metallocomplex (ZnTPPS4) sensitizers in the presence or absence of 2-hydroxypropyl-b-cyclodextrin (HP-b-CD) on G361human melanoma cells. Morphological changes in cell cultures have been evaluated using inversion fluorescent microscope and image analysis. Viability of cells was determined by means of molecular probes for fluorescence microscopy (LIVE/DEAD kit- double staining with Calcein AM and Ethidium Homodimer). The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorometric measurement with fluoroscan Ascent. We found that the most effective sensitizer is ZnTPPS4 bound to HPb-CD, since the IC50 value was 12.5 g/ml at the dose of light radiation of 10 J/cm2. # 2003 Elsevier Ltd. All rights reserved. Keywords: Sensitizers; Light; Phototoxicity; Photodynamic therapy

1. Introduction Photodynamic therapy (PDT) is a treatment for neoplastic disease that involves the selective destruction of tumors using light-activated sensitizer compounds that preferentially accumulate in target tissue areas (Sibata et al., 2001; Brown et al., 1999; Lui et al., 1993). The photochemical interactions of the sensitizer, light, and molecular oxygen produce cytotoxic singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radical and superoxide ion resulting in damage of organelles within malignant cells and leads to tumor ablation. The major sites of PDT damage are membranous organelles, such as mitochondria, lysosomes and plasma membrane (Dahle et al., 1999; Rogers et al., 1991; West et al., 1989). Efficiency of PDT is affected by Abbreviations: PDT, photodynamic therapy; TPPS4, meso-tetrakis(4-sulphonatophenyl)porphine; ZnTPPS4, zinc complex of TPPS4; HP-b-CD, 2-hydroxypropyl-b-cyclodextrin; CD, cyclodextrin; IC50, cytotoxic concentration resulting in a 50% reduction in cell viability. * Corresponding author. Tel.: +420-585632103; fax: +4205632167. E-mail address: [email protected] (H. Kola´rova´). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00094-8

various factors including photophysical properties of the sensitizer, wavelength of the activation light, depth of the light penetration in the biological tissue, tissue response on singlet oxygen, etc. (Moan and Berg, 1992; Zaidi et al., 1994) In our previous study (Mosinger et al., in press) we found that photodynamic sensitizers 5, 10, 15, 20-tetrakis(sulfonatopheny)porphyrin (TPPS4) and its zinc metallocomplex (ZnTPPS4) form 1:1 and/or 1:2 supramolecular complexes with native cyclodextrins (CD) and 2-hydroxypropyl cyclodextrins (HPCD) in aqueous neutral solutions. The formation of these assemblies causes a bathochromic shift of the porphyrin Soret band in the UV-VIS spectra and a red shift of the fluorescence emission bands. The binding constants span over three orders of magnitude from 8.1102 M 1 to 5.4105 M 1 (1.1106 M 2) depending on the size of the CD cavity and on the functionalization by adding 2-hydroxypropyl groups. The highest binding constants were obtained for HP-b-CD and HP-g-CD. The Nuclear Overhauser spectroscopy signals (ROESY) revealed hostguest type of interaction. The inclusion host–guest complexation does not influence the inherent photophysical

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Table 1 Values of IC50 (mg/ml) Sensitizer

TPPS4

TPPS4+HP-b-CD

ZnTPPS4

ZnTPPS4+HP-b-CD

IC50 (mg/ml) Without irradiation IC50 (mg/ml) With irradiation dose=10 J/cm2 Standard deviation n=9 IC50 (J/cm2) Without sensitizer

> 125 25 2.78 > 150

>125 18 2.13 >150

>125 15 1.82 >150

>125 12.5 1.98 >150

Significant influence of the levels of concentration at irradiation dose 10 J/cm2 was proved (P >0.05), n=9 for each concentration.

properties of the monomeric porphyrins such as the quantum yields of fluorescence, the triplet states, and the singlet oxygen formation. Due to the deaggregation effect of cyclodextrins, the inclusion complexes remain efficient supramolecular sensitizers of singlet oxygen even under conditions of extensive aggregation in aqueous solutions. In the present study we used TPPS4 and ZnTPPS4 as model sensitizers (Mosinger et al., 2000; Mosinger and Micka 1997). We report here the phototoxicity of these sensitizers in the presence or absence of 2-hydroxypropyl-b-cyclodextrin (HP-b-CD) and the influence of the sensitizers concentrations in combination with laser irradiation doses on photodamage of G361 human melanoma cells.

2. Materials and methods Twice washed trypsinized G361 human melanoma cells (ATTC, USA) were divided in the amount of 104 to each well (Dynatech plates 812, flat bottom) and filled in DMEM with 10% FCS in a total volume of 80 ml. After 24 h of cultivation at 37  C in 5% CO2 the sensitizer (20 ml) was added. Cells were cultivated with sensitizers at concentrations ranging from 0.1 to 125 mg/ml. The total volume of 100 ml (cells with additives) were cultivated for 24 h. The controls contained cells in the cultivation medium only. After 24 h of cultivation the cells were subsequently irradiated by a halogen lamp (24 V/250 W) at a dose of 0.5–150 J/cm2. The halogen lamp has continuous irradiance spectrum (from 360 to 2700 nm) with maximum in visible and near infrared region. The absorption maximum of sensitizers is at visible region (peaks at 420, 550 and 630 nm). Irradiance was measured by Radiometer RK 2500 (Meopta Prerov, Czech Republic). Morphological changes in cell cultures have been evaluated using inversion fluorescent microscope Olympus IX 70 and by image analysis Olympus MicroImage. Viability of cells was determined by means of molecular probes (Molecular Probes Europe BV) for fluorescence microscopy (double-staining with Calcein AM and Ethidium Homodimer). Fluorescent probes Calcein AM (excitation 495 nm, emission 515 nm) for the detection of live cells, Ethidium Homodimer (excitation 495 nm, emission 635 nm) for the detection of

dead cells were selected for the verification of cellular damage. The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorometric measurement with fluoroscan Ascent. The compounds were tested in quadruple in three independent experiments. The statistical evaluation of the results is based on analysis of the variance at twice sorting. Analysis of the variance was completed by Duncan test.

3. Results We report the influence of various concentrations of sensitizers in combination with light irradiation doses on the photodamage of G361 human melanoma cells. Viability studies have shown, that the optimum phototoxic effect observed in G361 melanoma cells was obtained in the presence of light dose of 10 J/cm2 and by concentration of ZnTPPS4 15 mg/ml, TPPS4 25 mg/ml, ZnTPPS4+HP-b-CD 12.5 mg/ml, TPPS4+HP-b-CD 18 mg/ml. These combinations of sensitizers concentrations and corresponding radiation doses were lethal for melanoma cells in tissue cultures. In Table 1 the IC50 values for all of the tested sensitizers are summarized. The values of IC 50 (50% inhibitory concentration) were estimated by graphical subtraction from doseresponse curve using software Microcal Origin (OriginLabTM Corporation, Northampton, USA). Dose– response curves were obtained as a dependence of relative ratio of live cells versus a concentration of the tested compound after 60 min incubation of the cells with

Fig. 1. Viability dependance of G361 cells on light dose—visible light only, without sensitizer. Viability of cells was not decreased under 90% after irradiation doses up to 150 J/cm2 .

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Fig. 2. Human melanoma cells G361 observed in transmited lightmicroscopy at 400 magnification. (a) Control G361 cells without irradiation. (b) Photodamaged G361 cells after PDT with ZnTPPS4 c=25 mg/ml, time of irradiation t=240 s, dose of light irradiation d=10 J/cm2. 

Calcein AM in a CO2-incubator at 37 C and 5% CO2. Doses of irradiation (0.5–150 J/cm2 ) were selected on the basis preliminary test which comfirmed that viability of cells was not decreased under 90% after irradiation doses up to 150 J/cm2 (Fig. 1). Microscopical study (Fig. 2) shows morphological changes in cell cultures after PDT treatment. Fig. 2a presents live control human meanoma cells G361 without irradiation. Fig. 2b shows photodamaged G361 cells after PDT with ZnTPPS4 c=25 mg/ml, time of irradiation t=240 s, dose of light irradiation d=10 J/cm2. All cells were dead. Fig. 3 shows fluorescence images after PDT treatment. Fig. 3a demonstrates detection of living cells by Calcein AM—green fluorescence on control cell lines. Fig. 3b shows detection of dead cells by Ethidium Homodimer—red fluorescence in photodamaged cells after PDT. Living and dead cells were counted by software Olympus Micro Image for each well. Calcein AM does not emit fluorescence until it is transformed into an intensively green fluorescing calcein by non-specific

Fig. 3. Detection of photodamage by fluorescence microscopy with fluorescent probes (double-staining with Calcein AM and Ethidium Homodimer) at 400 magnification. (a) Control living cells without irradiation—green fluorescence of Calcein AM. (b) Dead cells after PDT treatment red fluorescence of Ethidium Homodimer.

cytoplasmatic esterase. Ethidium Homodimer penetrates the cells through the damaged membrane, collocates to nucleic acids and produces fluorescence in the red field of spectrum in dead cells.

4. Discussion In this study we demonstrate in vitro phototoxicity using two model sensitizers. We have proved their low cytotoxic and high phototoxic effect in the visible region of spectrum. As these substances are expected to be used in photodynamic therapy, we have tried to find the sensitizer with the lowest cytotoxicity and highest phototoxicity possible. PDT requires three components present simultaneously for cytotoxicity: a sensitizer, light, and

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oxygen (Moor et al., 2000). The wavelength of light that activates the sensitizer used dictates the proper light spectrum as well as the depth of the treatment effect (Kolarova et al., 1998). PDT doses are associated with membrane photodamage and thus increased Ethidium Homodimer permeability (Fig. 2B). We found that the most effective sensitizer is ZnTPPS4 bound to HP-b-CD, since the IC50 value was 12.5 mg/ml at the dose of light radiation of 10 J/ cm2. According to our results ZnTPPS4 seems to be more phototoxic than TPPS4. G361 cells are sensitive to photodynamic damage by all of the tested sensitizers, but differences between them are not dramatic. In conclusion, TPPS4 and ZnTPPS4 in the supramolecular complex with HP-b-CD represent efficient sensitizers with high phototoxicity to G361 human melanoma cells. Acknowledgements The authors thank Olina Hustakova for assistance with tissue cultures. This work was supported by the grant project of Grant Agency No. 203/02/1483 Czech Republic and Ministry of Education No. MSM 153100008.

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