CONICET | Buscador de Institutos y Recursos Humanos

The outcome of the photodynamic therapy (PDT) depends on several factors such as genotype of cells, the nature of cell death, the environmental conditions, and PDT dosimetry, i.e., light intensity and photosensitizer concentration and its distribution at the affected tissue. In this contribution, we have carried out experiments on three dimensional HeLa cell spheroids (approximately 500 μm in size) to study the effect of dosimetry of the treatment. Meta-tetrahydroxy-phenyl chlorine (m-THCP) was employed as photosensitizer and the illumination was performed employing LED sources of 395 nm and 660 nm with an exposure time was in the range 4 - 12 minutes, and the drug concentration from 0.1 to 2.0 μg/ml. For asymmetric cell aggregates, light was directed from different angles. Light penetration was measures placing the spheroids between two coverslips and the transmitted light detected by a spectrometer. The outcome of the treatment was analyzed by confocal fluorescence microscopy after cell staining with vital staining. Intensity of acridine orange was evaluation from images to evaluate cell death. To asses the spheroid dynamics after illumination, cell death at different planes of the spheroid comparing, was compared at different times post treatment (3h, 12 h and 24 h) employing groups of spheroids illuminated with either 630 nm or 395 nm light, maintaining the fluence rate constant at 12,3 μW/cm2. Results indicate that the amount of death decreases in going from the surface of the spheroid closer to the light source to the lower planes. The fraction of dead cells follows a quasi-exponential relationship that correlates with light penetration and photosensitizer diffusion. Similar results have been reported in literature. This preliminary study indicates that the 395 nm LED light was more efficient than the 660 nm in agreement with 2D HeLa cells assays. Cell spheroids appear to be useful to obtain information about the dosimetry during PDT treatment.