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 × 10. 4. 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/cm. 2. -/-600 sec) and high output-/-short time (235 mW/cm. 2. -/-80 sec). The total energy density of both was 18.8 J/cm. 2. 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/cm. 2. 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

Photodynamic therapy (PDT) uses the strong cytotoxicity of singlet oxygen and hyperthermia produced by irradiating excitation light on a photosensitizer. The phototoxic effects of indocyanine green (ICG) and near-infrared light (NIR) have been studied in different types of cancer cells. Plasma proteins bind strongly to ICG, followed by rapid clearance by the liver, resulting in no tumor-selective accumulation after systemic administration. Kimura et al. have proposed using a novel nanoparticle labeled with ICG (ICG-lactosome) that has tumor selective accumulation owing to enhanced permeability and retention (EPR) effect. In this study, we investigated the efficacy of PDT using ICG-lactosome and NIR for a bone metastatic mouse model of breast cancer. Cells from the human breast cancer cell line, MDA-MB-231 were injected into the right tibia of 26 anesthetized BALB/C nu/nu mice at a concentration. The mice were then randomly divided into three groups: the PDT group (n = 9), the laser (laser irradiation only) group (n = 9), and the control group (n = 8). PDT was performed thrice (7, 21, 35 days after cell inoculation) following ICG-lactosome administration via the tail vein 24 hours before irradiation. The mice were percutaneously irradiated with an 810-nm medical diode laser for 10 min. In the laser group, mice were irradiated following saline administration 24 hours before irradiation. Radiographic analysis was performed for 49 days after cell inoculation. The area of osteolytic lesion was quantified. The right hind legs of 3 mice were amputated 24 hours after the third treatment. Histological analysis was performed using hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of sagittal sections. The data was analyzed using Tukey-Kramer post-hoc test. P-value of <0.05 was considered significant. X-ray on day 49 of the three groups are considered. The area of osteolytic lesion in the PDT group (7.9 ± 1.2 mm. 2. : mean ± SD) was significantly smaller than that of the control (11.4 ± 1.4 mm. 2. ) and laser (11.9 ± 1.2 mm. 2. ) groups. In histological findings, we observed many TUNEL-positive cells in the metastatic tissue 24 hours after PDT. In the control and laser groups, TUNEL-positive cells were occasionally observed. We have previously reported the effect of ICG-lactosome-enhanced PDT on the cytotoxicity of human breast cancer cells in vitroand on the delay of paralysis in a rat spinal metastasis model. In this study, we demonstrated the inhibitory effect of ICG-lactosome-enhanced PDT on bone destruction caused by human breast cancer cells in vivo. This PDT induced apoptosis and necrosis in the tumor cells. Intralesional resection is often performed for spinal metastases in an emergency. The residual tumor may regrow and cause neurological deficits. We believe that ICG-lactosome-enhanced PDT can decrease the rate of local recurrence through reduction of the residual tumor. PDT with ICG-lactosome and NIR had an inhibitory effect on the growth of bone metastasis of a human breast cancer