Polymersomes preparation
PMPC−PDPA Copolymers, useful for the assembling of the polymeric vesicles, were kindly provided by Prof. Giuseppe Battaglia and synthetized as described elsewhere (Fenaroli et al., 2020). PMPC−PDPA self-assembly of polymersomes was carried out using the thin film rehydration method. In particular, the polymers were first dissolved in a chloroform/methanol solution (2:1), in a 5% molar ratio between Cy5-labeled and unstained PMPC−PDPA polymers. The solvent was then evaporated, and the film was rehydrated with endotoxin/LPS-free Dulbecco’s PBS (Sigma-Aldrich) for a period of 4 weeks under vigorous stirring, to have a final polymer concentration of 10 mg/mL. After this period, the formed polymersomes were purified from the formed tubular structures, and only spherical nanoparticles were isolated, according to sucrose-based density gradient centrifugation (Robertson et al., 2016). These pre-purified samples were then further purified by size exclusion chromatography for isolating the Cy5-labelled nanovesicles and removing any unreacted free fluorescent dye.