In Vivo Experiments: Animals and Irradiation

All experimental animal protocols proceeded under supervision of the Ethics Committee (Faculty of Military Health Sciences, Hradec Kralove, Czechia). Female C57Bl/6 mice aged 12–14 weeks (Velaz, Unetice, Czechia) were kept in an air-conditioned room (22 ± 2°C and 50 ± 10% relative humidity, 12 h light/dark cycle) and allowed access to standard food and tap water ad libitum.

In conducting an experiment, mice were randomly divided into four groups. Prior to irradiation, all animals were anesthetized with a combination of Narketan (0.5 ml; Vetoquinol, Prague, Czechia), Rometar (0.16 ml; Bioveta, Ivanovice na Hane, Czechia), and physiological solution (2 ml; B. Braun Melsungen AG, Melsungen, Germany) by intramuscular injection. The first group (control, n = 18) was sham-treated and nonirradiated. Mice from the second group (n = 32) were only irradiated but without application of HANPs. The third and fourth groups (both n = 32) received HANPs of sizes 86.58 nm and 123.6 nm, respectively, by intratracheal instillation. The volume of HANPs was 50 µl in final concentration of 0.5 mg/ml and was instilled 30 min before irradiation. The anesthetized animals were kept in a Plexiglas box (VLA JEP, Hradec Kralove, Czechia) and received a single dose of irradiation (17 Gy, 0.30 Gy/min, 1 m) to the thoracic region. A local thoracic irradiation was performed in a jig. Head and abdomen were shielded with lead bricks 10 cm thick layer to reduce the dose to surrounding organs.