Abstract
Summary
Photodynamic therapy with ICG lactosome and near-infrared light has phototoxic effects on human breast cancer cells. With the same total energy, phototoxic effects depend on output of irradiation light rather than irradiation time.
Introduction
The phototoxic effects of indocyanine green (ICG) and near-infrared light have been studied in various fields. Plasma proteins bind strongly to ICG, which is followed by rapid clearance by the liver, resulting in no tumor selectivity after systemic administration. We have proposed a novel nanocarrier labeled with ICG (ICG lactosome) that has tumor selectivity due to its enhanced permeation and retention (EPR) effect. The aim of this study was to investigate in vitro phototoxic effects and to optimise the irradiation conditions by changing the output and time of near-infrared light as excitation light.
Materials and Methods
MDA-MB-231 human breast cancer cells were seeded (2 × 104 cells per well) into 96-well plates. The plates were divided (16 wells/treatment) into the following groups: control/untreated, only ICG lactosome administration (ICG lactosome), only laser irradiation (laser), and ICG lactosome administration plus laser irradiation (photodynamic therapy: PDT). Cells in the control, laser, and PDT groups were incubated in 100 μl medium for 24 h. Cells in the ICG lactosome group were incubated in 100μl medium containing 1 mg ICG lactosome for 24 h. The following day, laser group samples with 100 μl phosphate buffer solution (PBS) and PDT group samples with PBS containing 1 mg ICG lactosome were treated with laser irradiation using a near-infrared medical diode laser (λ = 810 ± 20 nm). Irradiation conditions were set to low output-/-long time (31 mW/cm2-/-600 sec) and high output-/-short time (235 mW/cm2-/-80 sec). The total energy density of both was 18.8 J/cm2. The media in these irradiated wells was replaced with fresh medium every 24 h post-irradiation. The control and ICG lactosome group wells received fresh medium every 24 h. Cells in all groups were incubated for 96 h post-treatment. Microscopic examination was performed, and cell viability was measured using a WST-1 assay every 24 h after treatment for 96 h. Mean absorbance in the WST-1 assay (an indicator of cell viability) was analyzed using the Tukey-Kramer test for comparison of multiple groups.
Results
Cell viability in the high output-/-short time PDT group was significantly lower than that in the low output-/-long time PDT group at 96 h after treatment. Cell viability in the two PDT groups was significantly lower than that in the other 3 groups at each time point. Irradiation increased the temperature by 25.5°C, 11.1°C, 8.1°C, and 7.1°C in the high output-/-short time PDT, low output-/-long time PDT, high output-/-short time laser, and low output-/-long time laser groups, respectively.
Discussion/Conclusion
PDT with ICG can penetrate deeper into tissue than that with Photofrin (the most widely used photosensitizer in clinical PDT), because ICG absorbs light at longer wavelengths than Photofrin. With the same total energy, inhibition of cell viability depended on irradiation output rather than irradiation time. It is reported that hyperthermia may contribute to the PDT effect if the surface irradiance exceeds 200 mW/cm2. We therefore believe that photodynamic and hyperthermal effects occurred in the high output-/-short time PDT group, and conclude that excitation light output rather than irradiation time may affect the photodynamic effect.
