*2.7. Erythrocyte Aggregation and Hemolysis*

Erythrocyte aggregation and hemolysis assays were performed for analyzing possible damaging effects of the polyplexes after incubation. Red blood cells from healthy mice were isolated by repeated washing steps with Ringer's solution and centrifugation for 5 min at 5,000 rpm until the solution became clear. After the last centrifugation step, the red blood cells were resuspended in 0.9% NaCl solution. For the determination of erythrocyte aggregation, 1 <sup>×</sup> 106 cells were diluted in 150 <sup>μ</sup>L 0.9% NaCl solution and incubated with the PPI-G4-Y/siRNA polyplexes or with 750 kDa branched PEI as positive control for 2 h at 37 ◦C. The cells were mounted onto coverslips and examined by bright field microscopy.

The hemolytic activity of the polyplexes was determined by incubating 50 μL polyplex solution with 50 <sup>μ</sup>L cell suspension containing 1.5 <sup>×</sup> <sup>10</sup><sup>7</sup> cells in saline for 1 h at 37 ◦C. Erythrocytes incubated with pure HN buffer served as negative control while cells lysed with 2% Triton X-100 were used as positive control (= 100% value).
