From Droplets to Fibers: Sequence-Encoded Phase Transitions of Synthetic Proteins for Oral Drug Delivery

Abstract

Proteins, the molecular building blocks of life, are genetically programmed to assemble into materials that regulate biology. Insights into the material properties of these assemblies have redefined the study of cellular organization and degenerative diseases, but we lack the tools to control these dynamic materials for biomedical applications. This dissertation covers a body of work in which we systematically study genetic fusions between synthetic polypeptides that form liquid droplets and short peptides that direct the formation of solid aggregates, with the goal of engineering proteins with dynamically-controlled material properties. We created a library of recombinant fusion proteins in which we varied the identities and the molecular architectures of aggregation-prone peptides within disordered scaffolds, then characterized their self-assembly behaviors and material properties. We generally found that increasing the percent composition of aggregation-prone peptides decreased thermal reversibility, which corresponded with a liquid-to-solid switch in material properties. This solidification transition at the molecular level resulted in microscale aggregation and hierarchical structures. We identified amyloid core peptides which could rationally change droplet solidification timescales based on pH, which we used to perform bottom-up fabrication of an unprecedented variety of morphologies. This allowed us to perform the first reported mechanistic interrogation of engineered protein assemblies as protective coatings for oral drug delivery. The largest spherical assemblies resulted in gastric acid survival times that match average stomach retention times in humans. Finally, we validated oral drug delivery in a preclinical mouse model of obesity using peptide weight loss drugs, finding that our encapsulated drug successfully enters the bloodstream after separating in the intestine, resulting in significant weight loss compared to controls. This work represents both a toolkit for controllable protein transitions and a mechanistic exploration of a platform technology for increasing oral drug delivery outcomes and patient compliance. Chapter 1, the introduction, explores protein interactions and summarizes the challenges and opportunities in the field of synthetic self-assembling protein materials. Chapter 2 covers early work in generating a library of proteins with multiple modes of self-assembly, which builds towards an understanding of how protein sequences and architectures determine phase transition behaviors. Chapter 3 introduces pH-responsive fiber-forming domains as motifs that endow a reversible liquid-solid switch into protein assemblies, with an emphasis on transitions for intestinal release of cargos. Chapter 4 explores the mechanistic basis for this liquid-solid switch, allowing us to exert exquisite control over the material properties and morphologies of assemblies. Chapter 5 builds on this concept with the interrogation of the factors that endow gastric acid survival in protein assemblies. In Chapter 6, we demonstrate the oral delivery of peptide drugs in preclinical models of obesity using the best performing proteins selected by the culmination of all the concepts and heuristics uncovered in this work. Finally, Chapter 7 discusses the implications, future steps, and translational considerations for this new class of protein materials and their drug delivery capabilities.