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To mimic organelles and cells and to construct next-generation therapeutics, asymmetric functionalization and location of proteins for artificial vesicles is thoroughly needed to emphasize the complex interplay of biological units and systems through spatially separated and spatiotemporal controlled actions, release, and communications. For the challenge of vesicle (= polymersome) construction, the membrane permeability and the location of the cargo are important key characteristics that determine their potential applications. Herein, an