In vitro toxicity testing of phthalocyanines on different cell lines using a continuous laser source
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Photodynamic therapy is a promising treatment for cancer. It involves the combination of a photosensitizer and light of an appropriate wavelength (laser source) to cause the destruction of cancer cells. Phthalocynanines are second–generation photosensitizers with enhanced photophysical and photochemical properties. In this in vitro study the effect of aluminium (AlTSPc) or zinc (ZnTSPc) tetrasulfophthalocyanines in its inactive and active state (laser induced) on melanoma (skin cancer cells), fibroblast (healthy normal skin cells) and keratinocyte (healthy normal skin cells) cells was evaluated. For each of the cell lines approximately 3 x 104 cells/ml were seeded onto 24-well cell culture plates and allowed to attach overnight, after which cells were treated with different concentrations of AlTSPc or ZnTSPc. The photosensitizers were synthesized at Rhodes University. After 2 hrs, cells were irradiated with a diode laser at a wavelength of 672 nm and a beam diameter of 1 cm. The laser power varied between 20-30 mW and the irradiation time was calculated to deliver a light dose of 4.5 J/cm2. Post-irradiated cells were incubated for 24 hrs before cell viability was measured using the CellTiter-BlueTM Viability Assay. Also, the efficacy of the light dose and laser source used for the killing of approximately 50% of the melanoma cancer cells were investigated. AlTSPc and ZnTSPc decreased cell viability of melanoma cancer cells to approximately 50% with photosensitizer concentrations of 40 μg/ml and 50 μg/ml respectively. These photosensitizer concentrations caused a slight decrease in the percentage cell viability of fibroblast and keratinocyte cells. Results for the dark toxicity assay showed that iii both photosensitizers in the presence of high concentrations (60 μg/ml – 100 μg/ml) showed cytotoxicity effects on melanoma cancer cells in their inactive state. This was not observed in fibroblast and keratinocyte cells treated under the same experimental conditions. The optimal AlTSPc and ZnTSPc concentrations in combination with the light dose of 4.5 J/cm2 was the most efficient in killing the melanoma cancer cells with reduced killing effects on healthy normal fibroblast and keratinocyte cells when compared to other light doses (2.5 J/cm2, 7.5 J/cm2 and 10 J/cm2). The irradiation of cells photosensitized with the optimal photosensitizer concentrations with a femtosecond laser using similar laser parameters to continuous wave laser experiments resulted in a reduction in the cell viability of healthy normal fibroblast and keratinocyte cells compared to melanoma cancer cells. The presence of DNA degradation on agarose gel, morphological changes like blebbing and ultrastructural changes like nucleus condensation indicated that photodynamic therapy treated melanoma cancer cells with the optimal concentrations of AlTSPc and ZnTSPc induced cell death via apoptosis. This concludes that low concentrations of AlTSPc and ZnTSPc activated with an appropriate laser source can be used to induce cell death in melanoma cancer cells. Both AlTSPc and ZnTSPc exhibit the potential to be used as a photosensitizer in photodynamic therapy for the treatment of melanoma cancer with the occurrence of minimal damage to surrounding healthy tissue.