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.