Erythrocyte hemolysis assay

The ability of free peptide, free polymer, and polyplex nanoparticles to disrupt lipid bilayer membranes in a pH-dependent manner was assessed by a hemolysis assay, as previously described [28]. Human blood from anonymous donors was obtained from Vanderbilt Technologies for Advanced Genomics (VANTAGE). Red blood cells (RBCs) were incubated with peptide, polymer, or polyplex nanoparticles at 5 μg/ml pPAA (2.5 μM) in 100 mM sodium phosphate buffer (supplemented with 150 mM NaCl) in the pH range of the endosomal processing pathway (7.4, 7.0, 6.6, 6.2, and 5.8). Extent of cell lysis (i.e., hemolytic activity) was determined spectrophotometrically by measuring the amount of hemoglobin released (abs = 541 nm) and normalized to a 100% lysis control (1% Triton X-100). Samples were run in quadruplicate.