*2.8. Blood Serum Markers and Immunostimulation*

Animal studies were performed according to the national regulations and approved by the local authorities (Landesdirektion Sachsen). The mice were kept in cages with rodent chow (ssniff, Soest, Germany) and water available *ad libitum*. Immunocompromised nude mice were maintained and worked with under sterile aseptic conditions.

For analyzing the immunostimulating cytokines TNF-α and INF-γ, PPI-G4-Y/siCtrl complexes containing 10 μg siRNA in 150 μL were intravenous (i.v.) injected twice within 24 h into healthy C57BL/6 mice. The blood was collected four hours after the last injection. Untreated mice served as negative control and mice treated with a single dose of 50 μg lipopolysaccharide (LPS from E.coli O111:B4; Sigma-Aldrich) were used as positive control. The serum levels of TNF-α and INF-γ were measured using ELISA kits (PreproTech, Hamburg, Germany) according the manufacturer's instructions.

For measuring the blood serum markers alanine-aminotransferase (ALAT), creatinine and urea, healthy nude mice were intraperitoneal (i.p.) and i.v. injected with complexes containing 10 μg siRNA as detailed in the figure. The mice were treated twice within 24 h and the blood was collected 72 h after the first injection. Untreated mice served as negative control. The serum was diluted 1:1 and analyzed using an AU480 (Beckman Coulter, Krefeld Germany).
