1 National Institute of Public Health, Šrobárova 48, Praha 10, Czech Republic

We have been studying some properties of the new generation of photosensitizers. We have chosen especially phthalocyanine ClAlPcS₂ and ClAlPcS₃. A semiconductor laser with output power of 21 mW and 675 nm was selected, because its wavelength corresponds to absorption maximum of phthalocyanines in the long-wave region of the visible part of electromagnetic spectra. To find optimum therapeutic doses for i. v. or local application, it was necessary to test the maximum non-toxic concentration of phthalocyanines and minimum phototoxic level related to laser irradiation in different model systems. Standard testing system for definition of the in vitro cytotoxicity and phototoxicity based on MCF7 (human breast adenocarcinoma), was developed. The optimum combination of phototoxic concentration and laser irradiation of wavelength 675 nm at a dose of 25 J/cm² was 1.8 m g/ml ClAlPcS₂ and 0.7 m g/ml ClAlPcS₃ respectively. Efficiency of PDT was affected by the following factors: absorption spectrum of the photosensitizer, wavelength of the activation light, depth of the light penetration in the tissue, and see tissue response to singlet oxygen.

Keywords: In vitro Photodynamic Therapy; Phthalocyanines; MCF7 cells; phototoxicity;

Photodynamic therapy (PDT) is a treatment that is used for destruction of certain tumours. The treatment is performed with dyes that generate reactive oxygen species in the presence of light and oxygen. Reactive oxygen species cause both cell death and damage to blood vessels thus contributing to tumour regression. Dyes such as hematoporfyrin derivates for this type of therapy are used (1). Disadvantage of the hematoporfyrin dyes is that they exist as a mixture of various, mostly unidentified compounds. Moreover, they absorb light at relatively short wavelengths that do not penetrate deeply into tissue. To circumvent these disadvantages other dyes are tested in PDT such as chlorins, phthalocyanines, etc.

Phthalocyanines are chemically pure compounds that absorb light of longer wavelengths than hematoporphyrin derivates with absorption maximum at about 680 nm (2, 3, 4, 5, 6). Phthalocyanines, belonging to a new generation of substances for PDT are chelated with a variety of metals, chiefly aluminium and zinc, while these diamagnetic metals enhance their phototoxicity (7). A ring substitution of the phthalocyanines with sulphonated groups will render them water soluble and affect the cellular uptake. Less sulphonated compounds, which are more lipophilic, show the best membrane - penetrating properties and highest biological activity (8). The kinetics are much more rapid than those of HPD, with high tumour to tissue ratios (8:1) reached after 1-3 hours. The phthalocyanines are eliminated much more rapidly, almost without fluorescence seen in the tumour after 24 hours (9).

In this paper we report on the optimal combination of maximum non-toxic concentration of phthalocyanines and minimum phototoxic level related to laser irradiation for MCF7 cells. The flourometric measurement with Fluoroscan Ascent was used for definition of in vitro cytotoxicity and phototoxicity. Morphological changes of the MCF7 cells were observed in an inversion fluorescent microscope Olympus IX 70.

Photosensitizers: Phthalocyanines ClAlPcS₂ and ClAlPcS₃ were synthesized by Jan Rakušan in the Research Institute for Organic Syntheses, Pardubice-Rybitví, Czech Republic

Source of light: Semiconductor laser Med 140 (Lasotronic, Ltd. l = 675 nm, 21 mW) was used as a source for invocation of the photodynamic effect.

Cells: MCF7 (Human breast adenocarcinoma) cells (obtained from Masaryk Memorial Cancer Institute, Brno-Czech Republic) were used as a standard testing system.

Phototoxicity of phthalocyanines for MCF7 cells. Twice washed tripsinized MCF7 cells were divided in amount 10⁴ to each well and filled in MEM with 10% FCS in a total volume of 0.1 mg/ml. After 24 hours of cultivation at 37 ^oC in 5% CO₂ the photosensitiser was added. The final concentration of phthalocyanine ranged from 0.1 m g/ml to 1540 m g/ml in a total volume 0.15 ml. MCF7 cells in mixture with photosensitizers were cultivated for 24 hours. Cytotoxicity of ClAlPcS₂ and ClAlPcS₃ without irradiation for MCF7 was tested. The values of IC₅₀ were obtained. Only the cells in the range from 0.1m g/ml to 10 m g/ml were subsequently irradiated by a laser of wavelength 675 nm at a dose from 10 J/cm² to 100 J/cm². The controls contained cells in a cultivation medium only. Morphological changes of the MCF7 cells during cultivation have been observed in an Olympus IX 70 inversion fluorescent microscope. The vitality of cells was evaluated 24 hours after irradiation. For identification of live and dead cells, the fluorescent probes ethidium homodimer and calcein were used. The previous results were also proved by flourometric measurement with Fluoroscan Ascent.

Photosensitizers with high absorption in the long-wave region of the visible spectra are suitable for application in photodynamic therapy (PDT). The phthalocyanines have the best absorption ability in a long-wave region of the spectrum as can be seen from the results of photosensitizer spectrum property studies (10). The absorption spectrum of ClAlPcS₂ phthalocyanine is shown in Fig.1

Fig.1 Absorption spectrum of phthalocyanines

MCF7 cells were irradiated by a laser before incubation without addition of photosensitizers. Laser irradiation of wavelength 675 nm at doses in the range from 10 J/cm² to 500 J/cm²was used. The vitality test proved that the vitality was not significantly influenced in the ranges of doses tested. Control measurements were in the range of 90% - 100%.

b) 4 m g/ml ClAlPcS₂ and laser irradiation 25 J/cm²