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

Beyond knockdown efficacy, biocompatibility is a major variable determining optimal complexes for transfection. Complexes based on PPI-G4-Y as well as those based on tyrosine-modified linear or branched PEIs proved to be highly biocompatible, as seen in cell viability assays in PC3 cells (Figure 4A). Notably, in particular transfection with PPI-G4-Y/siRNA complexes preserved cell viability by 100%. The absence of acute toxicity was also confirmed by LDH release assays, indicating no acute cell damage upon transfection with any of the tested complexes (Figure 4B). In line with this, no adverse effects on erythrocytes were observed, as shown here for PPI-G4-Y/ siRNA complexes. Erythrocytes incubated with PPI-G4-Y/siRNA complexes revealed no signs of aggregation, leaving them indistinguishable from their untreated counterparts (Figure 4C). Likewise, independent of complex amounts, no hemoglobine release from erythrocytes was detected (Figure 4D).

**Figure 4.** (**A**) Analysis of PC3 cell viability by WST-8 based measurement of viable cells upon transfection with various tyrosine-modified PPI- or PEI-based siRNA complexes at different amounts (indicated by siRNA amounts on the x-axis). Statistics analyze differences of the highest siRNA amounts (12 pmol) to untreated. (**B**) Assessment of acute cytotoxicity in PC3 cells by lactate dehydrogenase (LDH) into the medium. (**C**) Analysis of hemoglobin aggregation and (**D**) of hemoglobin release upon incubation of erythrocytes with PPI-G4-Y/siRNA complexes.
