Evaluation of the Sonosensitizing Activities of 5-Aminolevulinic Acid and Sn(IV) Chlorin e6 in Tumor-bearing Chick Embryos

Materials and Methods

Chemicals. 5-ALA hydrochloride was a gift from SBI Pharmaceuticals Co., Ltd. (Tokyo, Japan). SnCe6 was synthesized in our laboratory according to a patented method (15) by using chlorin e6 (Funakoshi Co., Ltd., Tokyo, Japan) as a starting material. Triton X-100 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Meylon was obtained from Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan).

Cell culture. Mouse mammary EMT6/KU tumor cells (supplied by Dr. Shin-ichiro Masunaga, Kyoto University, Kyoto, Japan) were maintained in Eagle's minimum essential medium (Sigma-Aldrich, Tokyo, Japan) supplemented with 10% fetal bovine serum (JR Scientific, Inc., Woodland, CA, USA). Cells were cultured in a 5% CO₂ in a fully-humidified atmosphere at 37°C.

Inoculation of EMT6/KU cells onto chorioallantoic membrane. Fertilized chicken eggs were purchased from Goto Hatchery, Inc. (Gifu, Japan). The first day of incubation was considered day 1. The fertilized chicken eggs were incubated in a humidified incubator at 37.6°C until day 11. On day 11, eggs were candled using halogen light to mark Y-shaped blood vessels on the chorioallantoic membranes (CAM). Over each of the identified blood vessels, a 1.5×1.5 cm square window was cut into the eggshell by using a grinder. After the eggshell and membrane were removed, the windows were sealed with polyurethane film (Opsite, Smith & Nephew, London, UK). Teflon rings were placed on the Y-shaped blood vessels and 2.5×10⁵ EMT6/KU cells/embryo were transferred to the Teflon ring. The window was resealed with transparent polyurethane film (3M Tegaderm™, St. Paul, MN) and the eggs were incubated at 37.6°C for 48 h (16). The Teflon rings were removed on day 13 and the eggs were incubated until day 15.

Intravenous administration of sonosensitizers in tumor-bearing chick embryos. On day 15, the eggs prepared with the above section were candled using halogen light to mark a thin blood vessel on the CAM. Rectangular 0.5×2 cm windows were cut into the eggshells over the thickest blood vessels. To visualize the blood vessels under the membrane, liquid paraffin was dropped onto the eggshell membrane. 5-ALA and SnCe6 were dissolved in physiological saline and meylon, respectively. Intravenous (i.v.) administration of 0.1 ml of each sensitizer was performed using a 30-gauge needle (16). After the i.v. administration, tumor, liver, and blood samples were collected at intervals up to 24 h. Serum was prepared from blood by using centrifugation at 3,000 rpm (735 × g) for 10 min. Tumors and livers were rinsed twice in physiological saline and stored at −80°C until they were analyzed.

Analysis of sonosensitizers. Tumor and liver tissues stored at −80°C were thawed at room temperature, weighed, and then homogenized in a solution of 2% Triton X-100 using a Handy micro-homogenizer Physcotron (Microtec Co., Ltd., Chiba, Japan) at 24,000 rpm for 10 s. After homogenization, the sample solutions were centrifuged at 15,000 rpm (160,000 × g) for 15 min and their supernatants were collected in new microcentrifuge tubes. PpIX and SnCe6 in the supernatants were quantified by measuring the fluorescence intensity using a LLS-405 VIS LED light source for excitation and a SEC2000-VIS/NIR fluorescence detector (BAS Inc., Tokyo, Japan). The excitation and emission wavelengths of PpIX were 400 nm and 632 nm, respectively, whereas the excitation and emission wavelengths of SnCe6 were 400 nm and 641 nm, respectively. The concentrations of PpIX and SnCe6 in the samples were determined from standard curves.

In vivo ultrasound irradiation. On day 15, tumor-bearing chick embryos received ultrasound irradiation 10 min and 5 h after SnCe6 and 5-ALA administration, respectively. Irradiation was performed directly on the solid tumors on the CAM by using a Sonitron GTS equipped a 12 mm transducer (Nepagene Co., Ltd., Chiba, Japan) under non-thermal conditions (986 kHz, 2.5 W, 60% pulse, 10 min). On day 18, the chick embryos were sacrificed and tumors were dissected from the CAMs.

Statistical analysis. Data are expressed as the mean±standard deviation. The differences between the results of the independent experiments were statistically analyzed using Student's t-test. A p-value <0.05 was considered statistically significant. Thompson's rejection tests were performed for individual values.