In vivo biodistribution of cold Pt-species and quantification of Pt accumulation

The Pt-BP and Pt(NO₃)₂(en) complexes were administered intravenously in the tail vein of C57BL/6N mice (n = 5 per each platinum complex). Sterile saline solution (0.9% NaCl) was used to dissolve platinum complexes. The concentration of injected Pt-BP or Pt(NO₃)₂(en) solutions was 2.5 mM platinum, calculated based on the maximum tolerated dose for cisplatin of 6 mg per kg body weight of the mice³⁷. The mice were euthanized with CO₂ 24 h after injection, after which blood (approximately 600 mg per mouse), liver, spleen, kidneys, heart, lungs, and bones (femur, humerus, tibia, and spine) were harvested. Approximately half of the tissues were prepared for inductively coupled plasma-mass spectrometry (ICP-MS) analysis by digestion in 65% (v/v) nitric acid at 75 °C for approximately three days until the tissues were digested completely. Each tissue was cut into three samples and the samples were weighed prior to digestion in nitric acid. The digested solutions were diluted with up to 6 ml of Milli-Q water to obtain 2% (v/v) nitric acid in order to measure platinum concentrations by ICP-MS (X series I, Thermo Electron Corporation). The detection limit for determining platinum concentration with ICP-MS was 1 ppb. The standard solutions were prepared from 1000 mg·1⁻¹ platinum ICP standard Certipur (1.70341.0100, Merck) ranging from 1 ppb to 2500 ppb. The measured isotopes for platinum were ¹⁹⁴Pt, ¹⁹⁵Pt, ¹⁹⁶Pt, and ¹⁹⁸Pt. Furthermore, scandium (⁴⁵Sc) was added as an internal standard, which was prepared with 1000 mg·1⁻¹ scandium standard Certipur (1.19513.0100, Merck) to correct for matrix effects and long-term fluctuations of the measurement signal. The platinum concentrations in the various tissues were calculated relative to 1 mg of the specific tissue and represented as ng Pt/mg_tissue. Three replicates of Pt-BP and Pt(NO₃)₂(en) solutions with volumes and platinum concentrations identical to the solutions injected to the mice (200 µl and 2.5 mM platinum, respectively) were also analyzed for their platinum content using ICP-MS as control. The percentage of injected dose (%ID) of Pt-BP and Pt(NO₃)₂(en) in each mouse tissue 24 h after injection was calculated by dividing the total amount of platinum per tissue through the total amount of platinum detected in the control solutions⁵².