CONICET | Buscador de Institutos y Recursos Humanos

The outcome of the photodynamic therapy (PDT) depends on several factors suchas genotype of cells, the nature of cell death, the environmental conditions, andPDT dosimetry, i.e., light intensity and photosensitizer concentration and itsdistribution at the affected tissue.In this contribution, we have carried out experiments on three dimensional HeLacell spheroids (approximately 500 μm in size) to study the effect of dosimetry ofthe treatment. Meta-tetrahydroxy-phenyl chlorine (m-THCP) was employed asphotosensitizer and the illumination was performed employing LED sources of 395nm and 660 nm with an exposure time was in the range 4 - 12 minutes, and thedrug concentration from 0.1 to 2.0 μg/ml. For asymmetric cell aggregates, lightwas directed from different angles. Light penetration was measures placing thespheroids between two coverslips and the transmitted light detected by aspectrometer. The outcome of the treatment was analyzed by confocalfluorescence microscopy after cell staining with vital staining. Intensity of acridineorange was evaluation from images to evaluate cell death. To asses the spheroiddynamics after illumination, cell death at different planes of the spheroidcomparing, 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/cm 2 .Results indicate that the amount of death decreases in going from the surface ofthe spheroid closer to the light source to the lower planes. The fraction of deadcells follows a quasi-exponential relationship that correlates with light penetrationand photosensitizer diffusion. Similar results have been reported in literature. Thispreliminary study indicates that the 395 nm LED light was more efficient than the660 nm in agreement with 2D HeLa cells assays. Cell spheroids appear to beuseful to obtain information about the dosimetry during PDT treatment.

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