Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres

), to form BCNs using the rapid and scalable technique of flash nanoprecipitation (FNP). Dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryoTEM) verified low polydispersity and the formation of bicontinuous structures with internal aqueous channels, respectively. Small-angle X-ray scattering (SAXS) confirmed a primitive cubic (Im3m) internal organization for BCNs assembled by FNP. Both hydrophobic and hydrophilic molecules were effectively loaded into BCNs via FNP, and encapsulated payloads were found to release in controlled manner in aqueous solutions. Due to the oxidation-sensitivity of PPS, biologically relevant concentrations of reactive oxygen species could trigger payload release on demand. BCNs were found to be non-toxic and endocytosed by phagocytic cells. Furthermore, an in vitro functional assay showed BCNs co-loaded with antigen ovalbumin and adjuvant monophosphoryl lipid A (MPL) to promote peptide/MHCI surface presentation by dendritic cells, a critical step for vaccine formulations during immunization. In conclusion, FNP supports the facile and scalable assembly and loading of PEG-bl-PPS BCNs, making them an attractive nanoscale delivery vehicle for both hydrophilic and hydrophobic molecules.