Intracompartmental 3D Printing of Enzymatically Active Organelle Mimics

Introducing subcellular structures in artificial cells is a key step in mimicking the structure and role of organelles, which are instrumental in compartmentalizing cellular reaction networks. Despite the variety of strategies to include subcellular features within artificial cell models, achieving spatial and morphological control over these compartments remains challenging. In this study, we engineered 3D-printed subcellular compartments within terpolymer-stabilized coacervate-based artificial cells. Coacervate-forming charged polymers were functionalized with methacrylate moieties, enabling the fabrication of a variety of architectures within droplets through photoinitiated radical polymerization. The addition of a Ni-NTA functional methacrylate monomer to the coacervates led to its sequestration upon polymerization in these subcellular regions. As a result, the compartments were able to uptake and concentrate His₆-tagged mTurquoise and β-galactosidase protein cargo molecules, despite the increase in viscosity that was induced upon polymerization. Following this affinity-based interaction approach, we demonstrated the region-specific localization of an enzymatic reaction within the artificial cells.