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Abstract

Formation of lipid-based compartments is a distinguishing feature of eukaryotic life forms. These compartments play a crucial role in orchestrating independent and self-contained metabolic, signalling or synthesis processes. Moreover, cellderived lipid compartments, like extracellular vesicles (EVs), have been shown to be essential for intercellular signalling and are involved in a wide variety of disease states. Although attaining a holistic understanding of EV-based communication is a compelling goal, the extensive molecular and structural complexity of these vesicles as well as a lack of reliable EV isolation techniques, have impaired detailed mechanistic insights. Inspired by bottom-up synthetic biology principles, the central goal of my interdisciplinary research was the development of a bio-inspired EV model system, which serves as a platform to study EV-based intercellular signalling and empowers novel EV-inspired therapeutics. In this thesis, I present two major methodologies developed for the controlled high-throughput assembly of synthetic vesicles. First, I describe a droplet-based microfluidic approach for the production of giant unilamellar vesicles (GUVs) with wellcontrolled biophysical and biochemical properties. I report on systematic investigations of GUV interactions with living cells and present concepts on how fine-tuning of the vesicles surface characteristics can be applied for targeted cellular delivery of macromolecular cargos. Moreover, I show how these vesicles can be reconceptualised as synthetic organelles, functioning within living cells and providing them with synthetic functionalities. Based on these fundamental characterizations, in the second part of my thesis, I present a complementary and quantitative approach for the sequential bottom-up assembly of fully synthetic EVs (fsEVs). To exemplify the application of fsEVs for new therapeutic concepts, I show that they exert analogous functionalities to naturally occurring wound healing EVs. Furthermore, by combining the fsEV technology with whole-transcriptome analysis, I systematically decode the synergistic functionalities between individual EV components. This approach enabled me to perform an analytical dissection of the associated EV signalling processes mediated by tetraspanin proteins. Bioinspired and biocompatible synthetic compartments with precisely controllable biophysical and biochemical properties are desirable tools for a wide range of living and synthetic cells research. This study makes it tempting to view EV-like compartments in a broader perspective. For example, they have great application potential as on-demand drug delivery systems, paving the way for hitherto impossible approaches towards administration of advanced cargos such as microparticles, viruses or synthetic organelles. Moreover, I anticipate that the highly controlled assembly of fsEVs will provide a robust framework for innovative therapeutic applications of bottom-up assembled synthetic biological modules and will additionally allow for new insights into fundamental EVrelated principles that govern cellular communication.