*3.4. In Vivo Biocompatibility of siRNA Complexes Based on PPI-G4-Y- or Various Tyrosine-Modified PEIs*

Studies on the biocompatibility of nanoparticles based on tyrosine-modified PPI or PEIs were also extended towards their application in vivo. Administration routes were intravenous (i.v.) or, considering the previously observed high efficacy and systemic availability of PEI-based nanoparticles, intraperitoneal (i.p.) injection [50,55]. In the case of PEI/siRNA complexes, these two modes of administration had previously shown marked differences in biodistribution and bioavailability, and thus both were included. After two injections, blood samples were taken and analyzed for serum

markers. Levels of urea (Figure 5A) and creatinine (Figure 5B) after treatment were found identical to the untreated negative control, indicating the absence of nephrotoxic effects. For the assessment of hepatotoxicity, alanine-aminotransferase levels were determined. In some cases, minor increases in alanine-aminotransferase (ALAT) levels were detected, but did not reach statistical significance (Figure 5C). Finally, immune stimulation was measured as well. In this case, studies were restricted to i.v. administration since cytokine activation occurs in a narrow time window and thus requires immediate bioavailability of the whole sample. To cover a very short as well as a somewhat longer time range, two injections of PPI-G4-Y/siRNA complexes within 24 h were performed, prior to the determination of TNFα and IFNγ levels. While a profound increase in the levels of both cytokines was observed in the lipopolysaccharide (LPS) positive control group, treatment with PPI-G4-Y/siRNA complexes led to no (TNFα) and or a slight, statistically not significant (IFNγ) alteration in serum levels (Figure 5D). In line with this, no alterations in animal appearance or behavior were observed throughout the experiments.

**Figure 5.** Determination of serum levels of (**A**) urea, (**B**) creatinine and (**C**) alanine-aminotransferase (ALAT) levels upon intraperitoneal (i.p.) or intravenous (i.v.) injection of mice with the tyrosine-modified PPI- or PEI-based siRNA complexes indicated on the x-axis. (**D**) Measurement of TNFα (left) and INFγ serum levels (right) upon i.v. injection of PPI-G4-Y/siRNA complexes.
