Arc 7010b

Solvents and reagents were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo Chemical Industry, Co., Ltd. (Tokyo, Japan), Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), Kanto Chemical, Co., Inc. (Tokyo, Japan), Merck (Darmstadt, Germany), and Chemescene (Monmouth Junction, NJ, USA), and used as provided. CO-1686 was purchased from AdooQ BioScience (Irvine, Canada). Gefitinib was purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Thin-layer chromatography (TLC) was performed on silica plates 60 F₂₅₄ (Merck, Darmstadt, Germany).
Nuclear magnetic resonance (NMR) spectra were recorded on a JNM-ECS400 (400 MHz) or JNM-ECA600 (600 MHz) spectrometer (JEOL Ltd., Tokyo, Japan). The mass spectrometry was performed on a JMS-T700 (JEOL Ltd., Tokyo, Japan) on fast atom bombardment mass spectrometry (FAB-MS). HPLC analyses and purification were carried out on a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan). The optical density in WST-8 assays were measured on an Infinite^® F200 Pro microplate reader (TECAN, Männedorf, Switzerland). Radioactivity was determined using an auto gamma counter ARC-7010B (Hitachi, Ltd., Tokyo, Japan).

Zn-InsP6 was prepared by the method previously reported with slight modification^{9 (link)}. Zn-InsP6 (Zn : InsP6 = 2 : 1) was used for both in vitro and in vivo experiments. This in vitro adsorption experiment was performed according to our previous study^{8 (link)}. Namely, Chlorella (10, 30, or 100 mg) or Zn-InsP6 (1, 3, 10, or 30 mg) was suspended and ²²³Ra (925 Bq) was added in 1 mL of the first test solution (artificial gastric juice, pH 1.2) or the second test solution (artificial intestinal juice, pH 6.8) defined in the Japanese Pharmacopoeia. After shaking the suspension at 1,000 rpm at 37ºC for 1 h using a shaking incubator (SI-300C; AS ONE Corp., Osaka, Japan), the samples were centrifuged at 10,000 g at room temperature for 10 min. The radioactivity of the supernatant was measured using an auto-well gamma counter (ARC-7010B; Hitachi Ltd., Tokyo, Japan) and the counts were corrected for background radiation. A window from 50 to 300 keV was used for the counting. The measurement time of each sample was set to 1 min. Control experiments were performed using the same procedure but without Chlorella or Zn-InsP6. The binding ratios were determined as follows:
Binding ratio to Chlorella or Zn-InsP6 (%) = [1 − (radioactivity of the supernatant of each sample) / (radioactivity of the supernatant of the respective control)] × 100.

DOX was purchased from LC Laboratories (Woburn, MA, USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEG-DSPE) were purchased from NOF Corporation (Tokyo, Japan). Cholesterol (Chol) was purchased from Sigma Aldrich (St. Louis, MO, USA). [¹²⁵I]Sodium iodide (644 GBq/mg) was purchased from PerkinElmer (Waltham, MA, USA). Other reagents were of reagent grade and used as received. Thin layer chromatography (TLC) was performed on silica plates 60 F254 (Merck, Darmstadt, Germany). Nuclear magnetic resonance (NMR) spectra were recorded on a JNM ECS400 (400 MHz) or JNM ECA600 (600 MHz) spectrometer (JEOL Ltd., Tokyo, Japan). The mass spectrometry was performed on a JMS T700 (JEOL Ltd.,) on electrospray ionization mass spectrometry (ESI-MS). HPLC analyses and purification were carried out on a Shimadzu Prominence HPLC system (Shimadzu Corp., Kyoto, Japan). Radioactivity was determined using an auto gamma counter ARC 7010B (Hitachi, Ltd., Tokyo, Japan). A colorectal adenocarcinoma cell line, Colon 26, was obtained from Cell Resource Centre for Biomedical Research in Tohoku University.