Delivery of Peptide Coacervates to Form Stable Interaction Hubs in Cells

Cells contain membrane-bound and membraneless organelles that operate as spatially distinct biochemical niches. However, these subcellular reaction centers lose fidelity with aging and as a result of disease. A grand challenge for biomedicine is restoring or augmenting cellular functionalities. Although commonly tackled by gene replacement therapy, an excited new strategy is the delivery of protein-based materials that can directly interact with and alter biological networks inside a cell. In this study we sought to develop long-lasting materials capable of cellular uptake and incorporation, akin to an artificial organelle or intracellular interaction hub. Drawing inspiration from protein-based membranelles organelles, we developed a new delivery method to transplant micron size peptide-based compartments into living cells. We determined conditions to form large stable coacervates that are efficiently taken up by a variety of useful cell types and demonstrate their intracellular stability over time. We developed tools to enhance the extent and spatial organization of cargo loading into these coacervates, including co-assembly of nanobodies that selectively bind to targets of interest. Combining them together, we demonstrate successful targeting of GFP protein inside cells. These results represent an important first step toward the development of deliverable synthetic organelles that can be fabricated in vitro and taken up by cells for applications in cell engineering and regenerative medicine.