Characterization and Optimization of Vesicle Properties in bioPISA: from Size Distribution to Post‐Assembly Loading

Abstract This study investigates the formation and properties of vesicles produced via biocatalytic Polymerization‐Induced Self‐Assembly (bioPISA) as artificial cells. Methods for achieving size uniformity, including gentle centrifugation and sucrose gradient centrifugation, are explored, and the effects of stirring speed on vesicle morphology is investigated. The internal structure of the vesicles, characterized by a polymer‐rich matrix, is analyzed using fluorescence correlation spectroscopy (FCS). Additionally, the feasibility of loading macromolecules into pre‐formed vesicles is demonstrated using electroporation, and a fluorescent protein as well as enzymes for a cascade reaction were sucesfully incorporated into the fully assembled polymersomes. These findings provide a foundation for developing enzyme‐synthesized polymeric vesicles with controlled morphologies for various applications, e.g., in synthetic biology.