General
Natural Ir powder (99.9%, d50 = 22.560 µm [median size]) was purchased from Furuya Metal (Tokyo, Japan), and sodium peroxide (95%) was purchased from Hayashi Pure Chemical Industry (Osaka, Japan). Ascorbic acid injection (500 mg/2 ml) was purchased from Fuso Pharmaceutical Industry (Osaka, Japan). Other chemicals and reagents were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan), Tokyo Chemical Industry (Tokyo, Japan), Kanto Chemical (Tokyo, Japan), Otsuka Pharmaceutical Factory (Tokyo, Japan), or Sigma-Aldrich (St. Louis, MO, USA), and were used in experiments without further purification. Milli-Q ultrapure water was used for dilution in all experiments.
HPGe γ-ray spectrometry was used for radioactivity measurements. The HPGe detector (EGC 15–185-R, Eurisys Measures, Strasbourg, France) was coupled with a 4096 multi-channel analyzer (RZMCA, Laboratory Equipment, Ibaraki, Japan), and calibrated using a mixed (109Cd, 57Co, 139Ce, 51Cr, 85Sr, 137Cs, 54Mn, 88Y, and 60Co) standard source (Japan Radioisotope Association, Tokyo, Japan). The efficiencies of each chemical separation process were defined as the ratio of 191Pt radioactivity after separation vs. before separation.
The HPLC system (PU-4080i; MD-4010, Jasco, Tokyo, Japan) was equipped with a 200-µL (analysis) or 1-mL (preparative isolation) sample loop and a radiation detector (US-3000, Universal Giken, Kanagawa, Japan). The analysis of *PtⅡCl42- was performed using a Nucleosil SB anionic exchange column (Chemco Plus Scientific, Osaka, Japan) at a flow rate of 1.5 mL/min, and eluted with perchlorate solution (MeCN/H2O = 40/60 (v/v) containing 0.1 mol/L NaClO4 and 0.04 mol/L HClO4). The analysis of [*Pt]cisplatin was performed using a polymer-based aqueous size exclusion chromatography (SEC) column (OHpak SB-804 HQ, Shodex, Showa Denko, Tokyo, Japan) at a flow rate of 1.0 mL/min, eluted with saline solution. Non-radioactive reference samples, K2PtⅡCl4 prepared in 0.1 mol/L HCl (0.5 mg/mL), and non-radioactive cisplatin in saline solution (0.5 mg/mL) were also analyzed to identify respective retention times.
Preparation of n.c.a. *PtⅡCl42-
The preparation scheme is shown in Fig. 1; the details of this scheme are as follows. As described previously [35], 188, 189, 191Pt was produced via the natIr(p,xn)188, 189, 191Pt reaction with a 30-MeV proton beam for 2–3 h at a beam current of 9–10 μA, and the irradiated Ir target (Ir: 120 mg, Na2O2: 98 mg) was dissolved in 6 mol/L HCl (6 mL). After filtering the solution, a stock solution containing mostly IrⅣCl62- with trace amounts of *PtⅣCl62- was prepared. In a typical batch, about 660 (189Pt) + 142 (191Pt) + 6 (188Pt) MBq of *PtⅣCl62- was produced at EOB (81.7 ± 0.4% of 189Pt, 17.6 ± 0.6% of 191Pt, and 0.7 ± 0.2% of 188Pt) and was used in the experiments.
Ascorbic acid, a reducing agent, was added to the filtered solution to selectively reduce IrⅣCl62- (ascorbic acid injection/Ir solution = 0.15/6 [v/v]). During this procedure, the dark reddish-brown solution turned greenish-yellow, as IrⅣCl62- was selectively reduced to IrⅢCl63- whereas *PtⅣCl62- remained intact. After the reduction, the mixed solution was loaded into a TBP-resin column made by connecting three TBP-resin cartridges (2 mL cartridge, TrisKem International, Rennes, France), as *PtⅣCl62- was selectively extracted into the resin. The column was rinsed with 3 mol/L HCl (5 mL), and then water (6 mL) was used as an eluting agent. To reduce *PtⅣ to *PtⅡ, an ascorbic acid solution (water + ascorbic acid injection = 2 + 8 mL) was added to the collected elution (6 mL), yielding a crude *PtⅡCl42- solution. The HCl concentration of this mixed solution was estimated to be <1 mol/L.
The resultant crude *PtⅡCl42- solution (16 mL) was loaded onto a column of QMA (Φ15×40 mm, AccellPlus QMA, Waters, Milford, MA, USA) for further purification, and the column was rinsed with 0.01 mol/L HCl (12 mL). An aqueous solution containing 1.5 mol/L HCl and 0.02 mol/L KCl, was used as an eluting agent. The elution was fractionated (2 mL each, f1–12), and the fractions containing *PtⅡCl42- (f5–10) were collected based on their radioactivity. The volume of the collected solution was decreased by evaporation, and the *PtⅡCl42- product (a precursor of [*Pt]cisplatin) was prepared in HCl and KCl solution (< 300 μL).
Synthesis of n.c.a. [*Pt]cisplatin
The synthesis scheme is shown in Fig. 1. About 900 μL of 3 mol/L ammonia solution was added to the *PtⅡCl42- solution until the pH reached a value of 8–9, as determined using a pH meter (D-72LAB; 9618S-10D, HORIBA, Kyoto, Japan). The mixed solution was heated in hot water at 60°C for 10 min, and then cooled in ice water. To isolate [*Pt]cisplatin, preparative HPLC was performed using a polymer-based aqueous size exclusion chromatography (SEC) column (OHpak SB-2004, Shodex, Showa Denko, Tokyo, Japan) at a flow rate of 3.0 ml/min, eluted with physiological saline solution.