*2.3. Complex Preparation and Characterization*

For standard transfection studies, the cells were seeded at a density of 35,000 cells per well of a 24 well plate in 0.5 mL fully supplemented medium. The following day, the cells were transfected with the polyplexes. The polyplexes were prepared at a polymer/siRNA mass ratio of 2.5 unless indicated otherwise. For a 24 well plate format, 0.4 μg siRNA in 12.5 μL HN buffer (150 mM NaCl, 10 mM HEPES, pH 7.4) and 1 μg tyrosine-modified polymer in 12.5 μL HN buffer were diluted in two separate tubes. The polymer dilution was added to the siRNA dilution, thoroughly mixed and incubated at room temperature for 30 min. After adding the polyplexes to the cells, no further medium change was performed.

The hydrodynamic diameters and zeta potentials of the PPI-G4-Y/siRNA complexes were measured by dynamic light scattering (DLS) and phase analysis light scattering (PALS), using the Brookhaven ZetaPALS system (Brookhaven Instruments, Holtsville, NY, USA). Polyplexes containing 5 μg siRNA in 250 μL total volume were prepared as described above and diluted to 1.7 mL in ultrapure water. The data were analyzed using the manufacturer's software, applying the viscosity and refractive index of pure water at 25 ◦C. For size determination, polyplexes were measured in five runs, with a run duration of 1 min per experiment. Zeta potentials were analyzed in ten runs, with each run containing ten cycles using the Smoluchowski model.

To study the complexation efficacy, agarose gel electrophoresis was used. Briefly, 0.2 μg siRNA was complexed in a total volume of 25 μL as described above at the different polymer/siRNA mass ratios indicated in the figure. The polyplexes were mixed with 10× loading dye and run on a 2% (w/v) agarose gel containing ethidium bromide at 80 V in TAE running buffer (20 mM EDTA, 40 mM Tris, 20 mM acetic acid). Unbound siRNA bands were visualized under UV illumination.

For assessing the complex stability of PPI-G4-Y/siRNA complexes, mass ratio of 3.75 was used. Complexes containing 0.2 μg siRNA in 25 μL were incubated with increasing amounts of heparin, incubated for 30 min at room temperature and subjected to agarose gel electrophoresis as described above.

To analyze the influence of FCS on the knockdown efficacies, PPI-G4-Y/siRNA complexes were further incubated in the presence of various FCS concentrations and storage conditions (fresh: 1 h at RT, 3 d at RT or 3 d at 37 ◦C).
