http://ar.iiarjournals.org/content/25/3B/2299.full.pdf+html?sid=c4c40b4a-1bc8-4cbc-92a7 97789011723c
Abstract
Photodynamic therapy (PDT) is a new treatment modality for tumours. The photochemical interactions of photosensitizer, light and molecular oxygen produce singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radicals and superoxide ions.
The tumour is destroyed either by ROS reactive singlet oxygen species (type II mechanism) and free radical products (type I mechanism) generated in an oxygen energy transfer reaction. The resulting damage to organelles within malignant cells leads to tumor ablation.
The cellular effects include membrane damage, mitochondrial damage and DNA damage. This process is non-selective in principle, and tumour specificity depends on a relatively high accumulation of the sensitizer in tumour tissue after systemic administration, combined with directed illumination.
The mechanism of PDT cytotoxicity (phototoxicity) is not fully understood. We studied cellular uptake and the phototoxicity of meso-tetrakis(4- sulphonatophenyl) porphine (TPPS4) and ZnTPPS4, PdTPPS4 sensitizers in the presence or absence of 2 hydroxypropyl-‚ -cyclodextrin (HP-‚-CD) on G361 human melanoma cells.
Twice-washed trypsinized cells (ATTC, USA) were divided in the amount of 10(4) to each well (Dynatech plates 8 x 12, flat bottom) and filled in DMEM with 10% FCS in a total volume of 80 ÌL. After 24 hours of cultivation at 37ÆC in 5% CO2, the sensitizer (20 ÌL) was added.
Cells were cultivated with sensitizers at concentrations ranging from 0.1 to 125 mg/ml. The total volume of 100 ÌL (cells with additives) were cultivated for 24 hours. The controls contained cells in the cultivation medium only.
After 24 hours of cultivation, the cells were subsequently irradiated by a halogen lamp (24V/250W) at a dose of 0.5 to 150 J/cm2. The halogen lamp has a continuous irradiance spectrum (from 360 to 2,700 nm), with maximum in visible and near infrared region. The absorption maximum of sensitizers is in the visible region (peak at 420, 550 and 630 nm).
Irradiance was measured by Radiometer RK 2500 (Meopta Prerov, Czech Republic). Morphological changes in cells were evaluated using inversion fluorescent microscope and image analysis. The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorimetric measurement with fluoroscan Ascent (Labsystems).
The viability of cells was determined by means of molecular probes for fluorescence microscopy (LIVE/DEAD kit). Sensitizer uptake into tumour cells may vary depending on the metabolic state of individual cells.
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Results
Using fluorometric assay of cell lysates, showed that the maximum distribution of sensitizers occurs before that of sensitizers in the supramolecular complex with cyclodextrin. The distribution of the sensitizers fixed in cyclodextrin carriers is slower and probably more effective.
According to our results, ZnTPPS4 seems to be more phototoxic than TPPS4 and PdTPPS4. G361 cells are sensitive to photodynamic damage by all of the tested sensitizers.
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Conclusion
ZnTPPS4, PdTPPS4 and TPPS4 in the supramolecular complex with cyclodextrin represent efficient sensitizers with high phototoxicity to G361 human melanoma cells.
This work was supported by the grant project of Grant Agency No. 203/02/1483 Czech Republic and Ministry of Education No. MSM 15310000
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H. Kolarova 1, M. Huf 1, R. Bajgar 1, M. Strnad 1, and J. Mosinger 2,
1. Department of Medical Biophysics, Faculty of Medicine, Laboratory of Growth Regulators, Palacky University, Hnevotínská 3, 775 15 Olomouc;
2. Department of Inorganic Chemistry, Faculty of Sciences, Charles University in Prague, Faculty of Social and Health Studies, University of South Bohemia, Ceske Budejovice, Czech Republic e-mail: kol@tunw.upol.cz
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http://ar.iiarjournals.org/content/25/3B/2299.full.pdf+html?sid=c4c40b4a-1bc8-4cbc-92a7 97789011723c
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