4.5. Biodistribution in Mice

Female C57BL/6 mice (25 g) used for this study were procured from Charles River (Wilmington, MA, USA) and housed in the Institutional animal house under standard environmental conditions (23 ± 1 °C, 55 ± 5% humidity and 12/12 h, light/dark cycles) and maintained with free access to standard diet and water. To establish the preliminary toxicological analyses, 4 groups of 5 mice were injected intravenously with 200 µL of saline solution with or without MSN at a concentration of 40 mg kg⁻¹. (1) Control group (saline solution injection) (2) Native-Fe₃O₄@MSN (3) DMPC-Fe₃O₄@MSN (4) PEG-Fe₃O₄@MSN.

Four days after treatment, mice were sacrificed and organs, blood and urine were collected for histological analysis and biochemical assays. The blood samples with heparin were centrifuged at 1 300 rpm for 10 min. The plasma and urine were stored at −20 °C up to analysis. We measured plasma cytokines (TNF-α and IL-6) to assess the inflammatory reaction or systemic toxicity. Plasma TNF-α and IL-6 levels were quantified using commercial ELISA kits as described in the manufacturer’s protocol (R&D systems, Minneapolis, MN, USA). The evaluation of renal function was determined by measuring creatinine levels in plasma and in urine using colorimetric assay at 495 nm with alkaline picrate. Liver function was determined from alanine aminotransferase (ALT) activity. Plasma ALT activity was measured according to standard protocol (Infinity, Thermo Scientific, Waltham, MA, USA). Moreover, liver, spleen and kidney were fixed in 10% paraformaldehyde, embedded in paraffin and cut 5 µm tick sections in a microtome. Sections were mounted on glass slides. After staining with hematoxylin-eosin, the sections were examined and imaged under a light microscope.

Inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantify silicium distribution in the digested tissue samples (liver, kidney and spleen). 2–100 mg of biological samples (urine, blood, or lyophilized and crushed kidney, spleen or liver) were digested in HNO₃ (1 mL, 67%) for 1 h, and then HCl (1 mL, 34%) and of H₂O₂ (0.5 mL, 30%) for 1 h. The digestion was completed by microwave in Teflon vials, by using UltraWAVE single reaction chamber (Milestone, Shelton, AL, USA). Silicium content was further analysed using a NexION™ 300 ICP-MS instrument (PerkinElmer, Waltham, MA, USA), from the analytical platform facilities of CEREGE (Aix en Provence, France).