*2.4. Polyplex Characterization*

The hydrodynamic diameters and zeta potentials were measured by dynamic light scattering (DLS) and phase analysis light scattering (PALS) with a Brookhaven ZetaPALS system (Brookhaven Instruments, Holtsville, NY, USA). Polyplexes containing 5 μg siRNA in 250 μL were prepared as described above and diluted to 1.7 mL with millipore water. The data were analyzed using the manufacturer's software, with applying the viscosity and refractive index of pure water at 25 ◦C. For the 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. Additionally, polyplex sizes were analyzed by nanoparticle tracking (NTA) using a NanoSight LM10 (Malvern, Wiltshire, UK) equipped with a 640 nm sCMOS camera, software NTA 3.0, and a siRNA concentration of 1 μg/mL.

Complex stabilities were determined by a heparin displacement assay. The complexes based on the different tyrosine-modified PEIs were prepared at their optimal mass ratios as described above, prior to further incubation in FCS (50%, *v*/*v*) for 1 h. The complexes were then subjected to the polyanion heparin at different concentrations (units), as indicated in Figure S3C. A total of 25 μL of the mixture containing 0.2 μg siRNA was analyzed by 1.5% agarose gel electrophoresis.
