Photodynamic therapy in 2D and 3D human cervical carcinoma cell cultures employing LED light sources emitting at different wavelengths.

Light of different wavelengths can be used to obtain a more profitable outcome of photodynamic therapy (PDT), according to the absorption bands of the photosensitizer (PS). Low-grade cervical intraepithelial neoplasias (CINs) are superficial lesions that can be treated with light of shorter wavelength than red because a large light penetration depth in tissue is not necessary. We report a comparative investigation performed to evaluate the efficacy of light-emitting diodes (LEDs) of different wavelengths in the photodynamic treatment applied to both 2D and 3D HeLa cell spheroid cultures. The spheroids are utilized as a PDT dosage model, and cell viability is evaluated at different sections of the spheroids by confocal microscopy. Cells incubated with m-tetrahydroxyphenyl chlorin are illuminated with LED systems working in the low fluence range, emitting in the violet (390-415 nm), blue (440-470 nm), red (620-645 nm) and deep red (640-670 nm) regions of the light spectrum at various exposures times (t I) comprised between 0.5 and 30 min. PDT experiments performed on both 2D and 3D cell cultures indicate that the PDT treatment outcome is more efficient with violet light followed by red light. Dynamic data from the front displacement velocity of large 2D-quasi-radial colonies generated from cell spheroids adhered to the Petri dish bottom as well as the evolution of the 3D growth give further insight about the effect of PDT at each condition. Results from 3D cultures indicate that the penetration of the violet light is appropriate to kill HeLa cells several layers below, showing cell damage and death not only in the outer rim of the illuminated spheroids, where a PS accumulation exists, but also in the more internal region. Results indicate that violet LED light could be useful to treat CINs involving superficial dysplasia.

Photodynamic therapy of HeLa cell cultures by using LED or laser sources.

The photodynamic therapy (PDT) on HeLa cell cultures was performed utilizing a 637nm LED lamp with 1.06W power and m-tetrahydroxyphenyl chlorin (m-THPC) as photosensitizer and compared to a laser source emitting at 654nm with the same power. Intracellular placement of the photosensitizer and the effect of its concentration (CP), its absorption time (TA) and the illumination time (TI) were evaluated. It was observed that for CP>40µg/ml and TA>24h, m-THPC had toxicity on cells in culture, even in the absence of illumination. For the other tested concentrations, the cells remained viable if not subjected to illumination doses. No effect on cells was observed for CP<0.05µg/ml, TA=48h and TI=10min and they continued proliferating. For drug concentrations higher than 0.05µgml(-1), further deterioration is observed with increasing TA and TI. We evaluated the viability of the cells, before and after the treatment, and by supravital dyes, and phase contrast and fluorescence microscopies, evidence of different types of cell death was obtained. Tetrazolium dye assays after PDT during different times yielded similar results for the 637nm LED lamp with an illuminance three times greater than that of the 654nm laser source. Results demonstrate the feasibility of using a LED lamp as alternative to laser source. Here the main characteristic is not the light coherence but achieving a certain light fluence of the appropriate wavelength on cell cultures. We conclude that the efficacy was achieved satisfactorily and is essential for convenience, accessibility and safety.