Calcein leakage experiments

For leakage experiments, calcein (25 mM) was dissolved in 0.1 M Tris–HCl, pH 8.14 and the polymersomes were formed in the calcein solution according to the method described above. Polymersomes and free calcein were separated on a 140 mL SEC column packed with Sepharose 4B (GE Healthcare). Fifty microlitre of the purified polymersome dispersion were added to 50 μL of the protein solution in 96 well, chimney, black polystyrene assay plates (Greiner bio-one, Kremsmünster, Austria) immediately before fluorescence measurements. The sample fluorescence was measured for 12 h in an Infinite M200 microplate reader. Measurements were taken every 5 min with excitation and emission wavelengths of 485 and 515 nm, respectively. Negative controls were always included to differentiate between eGFP fluorescence and calcein fluorescence. The positive control included 3 % triton X-100 to disintegrate the polymersomes and force calcein release.