The endogenous photosensitizer 5-aminolevulinic acid (ALA) is a haem precursor and induces the synthesis of protoporphyrin IX (PpIX) in mitochondria-containing cells. Due to the slow conversion of porphyrins to haem, high levels of PPIX are found in the tissues, sufficient to produce a photodynamic effect following exposure to light. Since PpIX accumulates effectively in tumor cells, the use of ALA leads to a better photoselectivity than Photofrin. However, this selectivity has not been sufficiently studied. As far as we know there is just one study comparing the amount of accumulated PpIX in non-tumor and tumor cell lines. In this work we attempt to compare not just the production but also the accumulation and cytotoxicity of PpIX in non-tumor (VERO) versus tumor (Hep-2) cells induced by the use of ALA. The results have shown that both non-tumor and tumor cell lines produce the same amount of PpIX but just the tumor cells can accumulate PpIX. So, under illumination, only the tumor cells will be killed.
Photodynamic therapy (PDT) is a technique for inducing tissue damage with light irradiation of a drug selectively retained in malignant tissue in presence of oxygen. The same mechanisms responsible for PDT efficacy can destroy the sensitizer, a process called photobleaching. In this work, the photobleaching of Photogem (a hematoporphyrin derivative used in Brazil made in Russia) was induced to study phototoxicity of the photoproducts upon a tumor (HEp-2) and non-tumor (VERO) cell line. Photogem was previously irradiated at 514nm and 630 nm (50mW/cm2) for 120 min. The sensitizer photobleaching was monitored by fluorescence and absorption properties changes and photoproducts formation evidenced by the appearance of a new absorption band around 640nm. Irradiated and non-irradiated Photogem were incubated for 18h with the cells. After drug removal, cells were irradiated with LED at 630 nm with intensities of 13, 20 and 25mW/cm2 for different times. Then, the cells were incubated for 48 hours to determine the cells viability by the MTT method. The cells in the dark were used as control. The IC50 decreases as light intensity increases, being more pronounced for tumor cells. Previously irradiated Photogem needs one-hour irradiation for both cell lines to have the same IC50 value of non-irradiated Photogem, which are irradiated for 14 min in tumor cells and 25 min in non-tumor cells. The results suggest that the photoproducts of Photogem are less cytotoxic either in the dark or in the light, decreasing with increase of intensity. These results underline the importance of dosimetry in PDT.
Porphyrins can be degraded by light. Photogem is a photosensitizer used in PDT in Brazil. Photogem irradiation at 514 and 630 nm change its fluorescence and absorption due to photodegradation and photoproducts formation (new absorption band in 640 nm). Irradiated Photogem was incubated with tumor or non-tumor cells. The low toxicity suggests that the photoproducts of Photogem are less cytotoxic than the original formulation.