The biological activity of PSP and ISP were evaluated in contrast to that of PPa in A549 human lung adenocarcinoma cells. An important prerequisite that a photosensitizer should have is a low dark toxicity when it is not irradiated. We therefore measured the dark toxicity of PSP and ISP by means of an MTT assay when cells treated by those compounds and untreated control cells kept in dark without any irradiation. After 3 h and 24 h of incubation in the dark, percentage of viable cells in each sample was measured by MTT assay (Figure 1).

At low concentrations, namely less than 3 μM, cells were not affected by the treatment with all compounds, whereas at concentrations more than 3 μM, all compounds exhibited a significant cytotoxic effect in A549 cells. Moreover dark toxicity of ISP was higher than those of PSP and PPa at concentrations more than 3 μM after 3h and 24 h incubation whereas the toxic effect induced by PSP was very lower than that of PPa at concentration more than 3 μM after 3 h and 24 h. To distinguish their dark toxicity effect quantitatively, we have evaluated the IC₉₀ for each compound, which were 4.2 (ISP), 7.1 (PPa) 14.8 μM (PSP) after 3 h and, 4.1 (ISP), 5.3 (PPa) 7.2 μM (PSP) after 24 h, respectively. Determined IC₉₀ values of compounds confirming that PSP exhibited drastically less toxic effect than both of ISP and PPa after 3h and 24 h incubation time.

We measured the phototoxicity of PSP, ISP and PPa at concentrations of 1.25, 2.5, 5, 10 and 20 μM in A549 cells when cells treated by those compounds and untreated control cells were irradiated with a 2 Jcm⁻² light dose. The percentage of viable cells was evaluated by MTT assay at 3 h and 24 h incubation time after PDT with increasing concentrations of those compounds as shown in Figure 2.

Percentage of cell viability decreased with increasing concentration of agents for all photosensitizers. For 3 h incubation after PDT, ISP exhibited enhanced phototoxicity than starting material PPa at all concentrations whereas PSP showed decreased phototoxicity compared to PPa at all concentrations, except for 1.25 μM. In the 24 h incubation time case, the phototoxicity of ISP was higher than that of PSP at all concentrations, whereas PSP showed more phototoxicity than PPa at concentrations of more than 5 μM.

According to the dark and phototoxicity study of PSP and ISP comparing with PPa, PSP exhibited not only low dark toxicity but also high phototoxicity at concentrations of 1.25 μM, whereas ISP exhibited high phototoxicity for all concentrations, but showed slightly higher dark toxicity only for high concentrations. The increasements of dark and phototoxicity for ISP may be explained by specific properties of imidazole which is involved in anticancer and antibacterial medications [18–19].

The phototoxicity of those compounds was also examined by monitoring cell death at 3 and 24 h incubation after photoirradiation under microscopic observation. Figure 3 shows the micrographs of A549 cells death at the concentration of 2.5 × 10-6 M and at different points of time after PDT. No cell death was observed without the agent, whereas cell death was observed at increased concentration of PSP and ISP. The leakage of the cytoplasm became more significant with time course and blebs were formed on the cell surface, indicating injury of the cell.

Absorption spectra were measured in CH₂Cl₂ using a SCINCO S-3100 spectrophotometer. ¹H-NMR spectra (300 MHz) were measured in CDCl₃ solutions with TMS as internal standard using a UNITYplus-300 spectrometer (Busan Center of the Korean Basic Science Institute, Busan National University, Korea); chemical shifts are expressed in ppm relative to TMS (0.00). Silica gel 60A (230–400 mesh, Merck) was used for column chromatography. All reactions were monitored by thin-layer chromatography (TLC) using Merck 60 silica gel F₂₅₄ precoated (0.2 mm thickness) glass-backed sheets. All chemicals, including imidazole, piperazine, MeOH, CH₂Cl₂, KOH, THF, SO₄H₂ and 1,3,5-collidine were analytical grade. Phosphate buffered saline (PBS, Sigma-Aldrich), a microscope (Olympus, CK40–32 PH, Japan), ELISA-reader (BioTek, Synergy HT, USA), trypsin-EDTA solution, and incubator (37°C, 5% CO₂) were also used.

Dried Spirulina Pacifica alga (500 g) was refluxed in acetone (2 L) under nitrogen for two hours. The supernatant was filtered. The extraction and filtration process was repeated three times. The green filtrate was evaporated and treated with 5% SO₄H₂ in MeOH for 12.5 hours at room temperature in the dark under nitrogen. After that, reaction mixture was treated with CH₂Cl₂, rinsed with water, 10% aqueous NaCO3H solution, and again with water three times. The organic layer was separated and dried over anhydrous Na₂SO₄ and evaporated to dryness. The residue was purified by column chromatography on silica gel eluting with 2% acetone in CH₂Cl₂ and the product was re-crystallized from CH₂Cl₂-MeOH. Methyl pheophorbide-a was thus obtained in 0.4% yield.

To a solution of PPa (0.1 mmole) in MeOH/CH₂Cl₂ (3:1, 10 mL), a solution of piperazine (0.1 mmole) in MeOH/water (1:2, 20 mL) was added and the mixture stirred about 2 hours. The organic solvents were evaporated under vacuum and the resulting aqueous solution was filtered through a membrane (20 μm) and freeze dried to give the piperazinium salt of PPa. λ_max(CH₂Cl₂) 667.3 nm; δ_H : 9.42 (1 H, s, H-5), 9.32 (1 H, s, H-20), 8.51 (1 H, s, H-10), 8.01 (1 H, m, H-3¹), 7.20 (2H, s, H-NH₂-pip), 6.28 and 6.16 (2H, d, H-3²), 5.30 and 5.09 (2 H, d, H-15¹), 4.46 (1 H, q, H-18), 4.26 (1 H, d H-17), 3.75 (2H, q, H-8¹), 3.60 (3 H, s, H-12), 3.51-3.41 (8H, m, H-CH₂-pip), 3.36 (3 H, s, H-1¹), 3.24 (3 H, s, H-7¹), 2.7 (2 H, m, H-17¹), 2.62 (2 H, m, H-17²), 2.08 (1H, br, NH-pip), 1.78 (3 H, d, H-18¹), 1.67 (3 H, t, H-8², J = 7.6 Hz, 7² Me), 0.88 (1 H, br, H-NH); -1.72 (1H, br, H-NH).

To a solution of PPa (0.1 mmole) in MeOH/CH₂Cl₂ (3:1, 10 mL), a solution of imidazole (0.1 mmole) in MeOH/water (1:2, 20 mL) was added and the mixture stirred about 2 hours. The organic solvents were then evaporated under vacuum. The resulting aqueous solution was filtered through a membrane (20 μm) and freeze dried to give the imidazolium salt of PPa. λ_max(CH₂Cl₂)/nm 667.1 nm; δ_h : 9.20 (1 H, s, H-5), 9.14 (1 H, s, H-20), 8.42 (1 H, s, H-10), 8.11 (2H, s, H-NH-imi), 8.01 (1 H, m, H-3¹ ), 7.66 (1H, s, H²-imi), 7.03 (2H, s, H⁴ and H⁵ -imi), 6.19 and 6.08 (2H, d, H-3²), 5.28 and 5.03 (2 H, d, H-15¹), 4.41 (1 H, q, H-18), 4.19 (1 H, d H-17), 3.72 (2H, q, H-8¹), 3.45 (3 H, s, H-12), 3.27 (3 H, s, H-1¹), 3.08 (3 H, s, H-7¹), 2.64 (2 H, m, H-17¹), 2.37 (2 H, m, H-17²), 1.73 (3 H, d, H-18¹), 1.58 (3 H, t, H-8², J = 7.6 Hz, 7² Me), 0.87 (1 H, br, H-NH), -1.77 (1H, br, H-NH).

The cell line tested was A549 (human lung carcinoma cell), which was obtained from the cell line bank at Seoul National University’s Cancer Research Center (Korea) and grown in RPMI-1640 medium (Sigma-Aldrich) with 10% fetal bovine serum, glutamine, penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO₂ in air. The PDT was carried out using irradiation (BioSpec LED, Russia). The wavelength was set at an appropriate level depending on absorption maximum of the photosensitizer. Duration of the light irradiation, under PDT treatment, is calculated taking into account of the empirically found effective dose of light energy in J cm⁻².

Cells from each cell line were inoculated into a 96-well chamber slide at a volume of 100 μL (5 × 10⁴ cells/well) for stationary culture. Twenty-four h later, photosensitizer (2.5 μM, 100 μL/well) was then added. After a predetermined time, the photosensitizer solution was discarded and the culture was again washed (x 3) with physiological saline and medium added to a volume of 100 μL/well. The cultures were then subjected to LED irradiation at the distance of 20 cm for 10 min, and examined by optical microscopy 3 h and 24 h later to determine the morphologic changes induced, compared with the cultures not subjected to irradiation. The time course of the changes in survival rate after irradiation was observed.

Cells from each cell line were inoculated into a 96-well, flat-bottomed microplate at a volume of 100 μL (1 × 10⁵ cells/well) for stationary culture. Forty-eight hours later, the medium was removed, and the cultures were washed three times with physiologic saline. Amounts of photosensitizer solution (0.3, 0.6, 1.25, 2.5 and 5.0 μM) were then added at a volume of 100 μL/well, respectively. Twenty-four hours later, the photosensitizer solution was discarded, and the cultures were again washed three times with physiological saline and then medium was added to a volume of 100 μL/well. The cultures were then subjected to the irradiation (2 J cm⁻²) at the distance of 20 cm for 10 min, followed by an 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assay to evaluate their sensitivity to PDT. For the MTT assay, MTT solution (10 μL) was added to each cell-culture well and cultured in the incubator for 3 h. Detergent solution (TACS™, Trevigen, 200 μL) was added to the culture, shaken for 10 min, and the absorbance was measured with an ELISA-reader at 570 nm. Measurements were performed 3 h and 24 h after the irradiation. Each group consisted of three wells.